Pharmaceutical compositions comprising human interleukin-4 (IL-4)

ABSTRACT

A pharmaceutical composition comprising human Interleukin-4 (IL-4).

This is a divisional of application Ser. No. 06/843,958, filed on Mar.25, 1986 now U.S. Pat No. 5,552,304, which is a continuation-in-part ofU.S. Ser. No. 06/799,668, filed on Nov. 19, 1985, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to the application of recombinant DNAtechnology to elucidate the control mechanisms of the mammalian immuneresponse and, more particularly, to the isolation of nucleic acid clonescoding for polypeptides capable of exhibiting B-cell, T-cell, macrophageand mast cell stimulatory activity, as well as colony stimulating factorenhancing activity.

BACKGROUND OF THE INVENTION

Recombinant DNA technology refers generally to the technique ofintegrating genetic information from a donor source into vectors forsubsequent processing, such as through introduction into a host, wherebythe transferred genetic information is copied and/or expressed in thenew environment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host.

This technology has progressed extremely rapidly in recent years and avariety of exogenous proteins have been expressed in a variety of hosts,but obtaining any desired novel cDNA clone remains an uncertainty. Byway of example, some of the eukaryotic proteins produced by recombinantDNA technology include: proinsulin (Naber, S. et al., Gene 21:95-1041983!); interferons (Simon, L. et al., Proc. Natl. Acad. Sci. U.S.A.,80:2059-2062 1983! and Derynck, R. et al., Nucl. Acids Res. 1:1819-18371983!); growth hormone (Goeddel, D., et al., Nature 281:544-548 1979!);a human T-cell growth factor (Taniguichi, T. et al., Nature 302:305-310(1983)); and a granulocyte/macrophage cellular growth factor (G/M-CSF)(Miyatake, S. et al., EMBO J. 4:2561-2568 (1985)). These publicationsand other reference materials cited hereafter have been included toprovide additional details on the background of the pertinent art and,in particular instances, the practice of the invention, and are allincorporated herein by reference.)

For some time, it has been documented that the mammalian immune responseis based on a series of complex cellular interactions, called the"immune network." Recent research has provided new insights into theinner workings of this network. While it remains clear that much of theresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes and other cells, immunologistsnow generally hold the opinion that soluble proteins (e.g., theso-called "lymphokines") play a critical role in controlling thesecellular interactions. Thus, there is considerable interest in theisolation, characterization, and mechanisms of action of cell modulatoryfactors, an understanding of which should yield significantbreakthroughs in the diagnosis and therapy of numerous disease states.

Lymphokines apparently mediate cellular activities in a variety of ways.They have been shown to support the proliferation, growth and thedifferentiation of the pluripotential hematopoietic stem cells into thevast number of progenitors composing the diverse cellular lineagesresponsible for the immune response. These lineages often respond in adifferent manner when lymphokines are used in conjunction with otheragents.

Cell lineages important to the immune response include two classes oflymphocytes: B-cells, which can produce and secrete immunoglobulins(proteins with the capability of recognizing and binding to foreignmatter to effect its removal), and T-cells of various subsets thatsecrete lymphokines and induce or suppress the B-cells and some of theother cells (including other T-cells) making up the immune network.

Another important cell lineage is the mast cell (which has not beenpositively identified in all mammalian species)--a granule-containingconnective tissue cell located proximal to capillaries throughout thebody, with especially high concentrations in the lungs, skin, andgastrointestinal and genitourinary tracts. Mast cells play a centralrole in allergy-related disorders, particularly anaphylaxis as follows:when selected antigens crosslink one class of immunoglobulins bound toreceptors on the mast cell surface, the mast cell degranulates andreleases the mediators (e.g., histamine, serotonin, heparin, kinans,prostaglandins, etc.) which cause allergic reactions, e.g., anaphylaxis,as well as others.

Research to better understand (and thus potentially treattherapeutically) various immune disorders has been hampered by thegeneral inability to maintain in vitro cells of the immune system.Immunologists have discovered that culturing these cells can beaccomplished through the use of T-cell and other cell supernatants,which contain various growth factors, such as some of the lymphokines.

The detection, isolation and purification of these factors is extremelydifficult, being frequently complicated by the complexity of thesupernatants they are typically located in, the divergencies andcrossovers of activities of the various components in the mixtures, thesensitivity (or lack thereof) of the assays utilized to ascertain thefactors' properties, and the frequent similarity in the range ofmolecular weights and other characteristics of the factors.

Clarification of these issues requires additional structural data, e.g.,substantially full-length sequence analysis of the molecules inquestion. Protein sequencing offers, of course, a possible means tosolve the problem, but it is very difficult work experimentally andoften can provide neither completely accurate nor full-length amino acidsequences. Moreover, having the capability of making bulk quantities ofa polypeptide exhibiting activity on the cells of the immune system willgreatly facilitate the study of the biology of those and other cells,e.g., by minimizing the necessity of relying on lectin conditioned mediafor stimulating cell growth. Accurate and complete sequence data on anyimmune protein will also serve to simplify the search for otherimmunological factors. Finally, additional information on any lymphokinewill help in evaluating the roles of the various growth factors andcells of the immune network and thus provide insight into the entireimmune system--with the concomitant therapeutic benefits.

Thus, there exists a significant need for extensive nucleotide sequencedata on the DNAs coding for, and amino acid sequences of, proteinsexhibiting immune cell growth or stimulatory activity, as well as asimple and economic method of making substantial and essentially purequantities of such materials. The present invention fulfills theseneeds.

DESCRIPTION OF ADDITIONAL RELEVANT REFERENCES

Metcalf, D. "The Hematopoeitic Colony Stimulating Factors," Elsevier,Amsterdam (1984), provides an overview of research concerninglymphokines and various growth factors involved in the mammalian immuneresponse. Yung, Y.-P., et al., J. Immunol. 127:794 (1981) describe thepartial purification of the protein of approximately 35 kd exhibitingmast cell growth factor (MCGF) activity and its separation frominterleukin-2 (IL-2), also known as T-cell growth factor (TCGF). Nabel,G., et al., Nature 291:332 (1981) report an MCGF exhibiting a molecularweight of about 45 kd and a pI of about 6.0. Clark-Lewis, I. andSchrader, J., J. Immunol. 127:1941 (1981) describe a protein having mastcell like growth factor activity that exhibits a molecular weight ofabout 29 kd in phosphate-buffered saline and about 23 kd in 6M guanidinehydrochloride, with a pI of between about but of about 6-8 afterneuraminidase treatment. Murine IL-2 and interleukin-3 (IL-3) have beenpartially characterized biochemically by Gillis, S., et al., J. Immunol.124:1954-1962 (1980) and Ihle, J., et al., J. Immunol. 129:2431-2436(1982), respectively, with IL-2 having an apparent molecular weight(probably as a dimer) of about 30-35 kd and IL-3 having a molecularweight of about 28 kd. Human IL-2 apparently has a molecular weight ofabout 15 kd and is described by Gillis, S., et al., Immun. Rev.63:167-209 (1982). Comparison between IL-3 and MCGF activities of T-cellsupernatants have been reported by Yung Y. and Moore, M., Contemp. Top.Mol. Immunol. 10:147-179 (1985) and Rennick, D., et al., J. Immunol.134:910-919 (1985).

B-cell stimulatory factor (BSF-1) has been separated physically fromIL-2 and shown to have a molecular weight of about 11 kd and about 15kd, with pI values of 6.4-6.7 and 7.4, respectively, by Farrar, J., etal., J. Immunol. 131:1838-1842 (1982). Ohara, J. and Paul, W., Nature315:333-336 describe a monoclonal antibody allegedly specific for murineBSF-1 and molecular weights for BSF-1 of 14 kd and 19-20 kd with a pI of6.7. The existence of at least two distinct human B Cell growth factors(BCGFs) has been suggested by others (see, Kishimoto, T., Ann. Rev.Immunol., 3:133-157 1985!). Yoshizaki, K., et al. (J. Immunol.130:1241-1246 (1983) showed that an IL-2 dependent helper T-cell clone(d4) produced a 50 KD BCGF distinct from a 15-20 KD BCGF isolated fromPEA stimulated T-cells, and the two BCGF's showed a synergistic effecton the proliferation of anti-IgM-stimulated B cells. Also, a factorhaving synergistic colony stimulating factor activity and otheractivities was described by Kriegler, A., et al., Exp. Hematol.12:844-849 (1984).

SUMMARY OF THE INVENTION

The present invention provides cDNA clones coding for immune modulatingfactor (IMF) polypeptides capable of exhibiting a broad spectrum ofactivities on mammalian hematopoietic cells, including B-cells, T-cells,granulocytes, macrophages and mast cells. Nucleotide sequences for twocDNA's and putative amino acid sequences for the associated polypeptidesare shown in FIG. 1. The cDNA sequences can be integrated into variousvectors, which in turn can direct the synthesis of the correspondingpolypeptides in a variety of hosts, including eukaryotic cells, such asmammalian cells in culture.

More specifically, the invention provides a process for producingpolypeptides exhibiting the desired activities, the process comprisingthe steps of:

a) forming a vector comprising a nucleotide sequence coding for one ofthe polypeptides, wherein the nucleotide sequence is capable of beingexpressed by a host containing the vector;

b) incorporating the vector into the host; and

c) maintaining the host containing the vector under conditions suitablefor expression of the nucleotide sequence into said polypeptides.

Preferably, the cDNA sequences are derived from T-cell mRNA sequencescoding for the native polypeptides, and the host is an organism such asa eukaryotic, e.g., mammalian cell transfected or transformed with thevector. Further, the vector preferably comprises also a secondnucleotide sequence capable of controlling expression of the nucleotidesequence coding for the polypeptide. This second sequence can include apromoter sequence, one or more intron sequences and a polyadenylationsequence, to permit, respectively, transcription, splicing andpolyadenylation of the nucleotide sequences coding for the polypeptides.Particularly, when the host is a mammalian cell, such as a COS-7 monkeykidney cell (COS), the vector contains the promoter sequence of theSimian virus 40 (SV40) early region promoter and the polyadenylationsequence of the SV40 late region polyadenylation sequence.

The cDNA sequences of FIG. 1 are capable of hybridizing with other DNAsequences, such as DNA coding for other mammalian IMF's from cDNA orgenomic libraries. Some of the cDNA sequences may contain informationfor a leader sequence.

The polypeptides of the present invention can be used to stimulateB-cells and to enhance T-cell, granulocyte and mast cell growth. Whenused in association with other immune-reactive agents, the activities ofsuch agents are enhanced. Suitable pharmaceutical compositions can beprepared by adding the polypeptides, within or without other agents, totherapeutically compatible carriers.

Other features and advantages of the invention will become apparent fromthe following detailed description, which describes, in conjunction withthe accompanying drawings and by way of example, the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a mouse cDNA insert that contains a single open-readingframe consisting of 140 codons.

FIG. 1B shows a human IL-4 cDNA that contains 153 amino acid residues.

FIGS. 2A and 2B, depict a map of an expression vector and cDNA insert ofthe present invention. (A) General diagram of pcD 2A-E3, a vectorcarrying a functional cDNA insert of the present invention. The cDNAinsert extends from the GC tail to the AT tail and contains theindicated restriction endonuclease cleavage sites; the coding region isheavily shaded, and the noncoding regions are lightly shaded. Thedirection of transcription from the SV40 promoter is indicated by thearrow. (B) Restriction endonuclease cleavage map of the insert in clone2A-E3 is shown.

FIGS. 3A, 3B, 3C and 3D illustrate some of the biological activities ofsupernatant from COS cells transfected with a cDNA clone of the presentinvention. (A) TCGF activity was determined with HT-2 cells using acolorimetric assay. Samples: 1) 2A-E3 COS supernatant. 2) Cl.Lyl⁺ 2⁻ /9cell supernatant. 3) COS-IL-2. 4) Mock transfected COS supernatant. (B)MCGF activity was determined using MC/9 mast cells and a colorimetricassay. Samples: 1) 2A-E3 COS supernatant. 2) COS-IL-3. 3) Cl.Lyl⁺ 2⁻ /9supernatant. 4) Mock transfected COS supernatant. (C) Expression of Iaantigen on B-cells cultured in test samples was determined byfluorescent staining. 1) 2A-E3 COS supernatant. 2) Cl.Lyl⁺ 2⁻ /9 cellsupernatant. 3) Mock transfected COS supernatant. The fluorescence unitswere calculated by multiplying the percentage of positive cells in eachsample by the intensity of fluorescent staining. (D) IgE (left) and IgG₁(right) levels in supernatants of LPS stimulated B-cells cultured withtest samples are shown. 1) Medium only. 2) 20% COS mock. 3) 20% Cl.Lyl⁺2⁻ /9 plus 20% COS mock supernatant. 4) 20% 2A-E3 COS supernatant.Specific immunoglobulin production was determined using an isotypespecific ELISA assay.

FIGS. 4A, 4B and 4C illustrate some of the biological activities ofsupernatants from COS or L cells transfected with other cDNA clones ofthe present invention (isolates from a human cDNA library). (A) TCGFactivity was determined with JL-EBV cells using a colorimetric (MTT)assay. (B) TCGF activity was also determined with PBL-PHA T-cell blastsusing a colorimetric assay. (C) TCGF activity was further analyzed onPBL-PHA T-cell blasts using a ³ H!-thymidine incorporation assay.Samples:

Open circles, Human IL-2;

Open triangles, Mock COS supernatant;

Closed Squares, COS-Human (clone #46);

Open squares, COS-Human (clone #125, oligo(dG) deleted); and

Closed circles, L cell-Human (clone #125, oligo(dG) deleted).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In accordance with the present invention, cDNA clones are provided insubstantially pure form, which clone that code for IMF polypeptidescapable of exhibiting a broad activity spectrum on, for example, almostall hematopoietic cell lineages. The responses of each cell lineage canvary depending upon the environment, including the presence ofadditional lymphokines or other immune-reactive agents. After the cDNAsequences have been incorporated into replicable expression vectors, andthe vectors transfected into an appropriate host (e.g., a mammalian cellculture), the expressed polypeptide or polypeptides can allow theexpansion (usually species-specific) of T-cells, mast cells, and/orother receptive hematopoietic cell lineages (e.g., granulocytic) in vivoor in vitro.

The polypeptides produced in accordance with the present invention canexhibit synergistic activity in cells when the factors are combined withother immune reactive agents, such as increased mouse mast andgranulocytic cell growth in conjunction with mouse IL-3 and increasedproduction of granulocytic colonies when the polypeptides are presentwith granulocyte/macrophage colony stimulating factor (G/M-CSF) orgranulocyte colony stimulating factor (G-CSF). These polypeptides canalso induce Ia expression on resting B-cells, enhance IgG₁ and IgEsecretion by B-cells, and promote the entry into S phase of B-cellsactivated by anti-IgM antibodies (see, Howard, M. et al., Immunol. Rev.78:185 (1984), which is incorporated herein by reference). Moreover, thepolypeptides can induce Ia induction on macrophages while limiting themacrophages' factor-dependent growth.

For purposes of describing this invention, IMF polypeptides are definedas the expression products, with or without processing of the nucleotidesequences (and allelic variants thereof) depicted in FIG. 1 and those ofrelated mammalian genes. In accordance with the assays described herein,the activities of IMF polypeptides can include, but are not limited to,B-cell, T-cell, macrophage, and mast cell stimulation; and enhancementof colony stimulating factor activity in progenitor cells. Otheractivities include affinity for receptors on such cells and/or specificantibody preparations; antigenicity upon introduction into a foreignhost; and the like. The processing may include: cleavage of 5 to 50amino acids or more, typically 10-20 amino acids, from an internalfragment or from the amino or carboxy terminus of a polypeptide;disulfide bridging; glycosylation; and other protein processing wellknown to those skilled in the art.

Two exemplary, putative amino acid sequences based on mouse-andhuman-isolated nucleotide sequences of the present invention are shownin FIG. 1A and 1B. FIG. 1A shows a mouse cDNA insert that contains asingle open-reading frame consisting of 140 codons. Downstream of theputative initiation codon is a region rich in hydrophobic amino acids.It is likely, therefore, that the mature native form of secretedpolypeptide begins with a histidine residue, and the preceding 20 aminoacids constitute a leader region, which is subject to removal byproteolytic processing. Thus, in one embodiment, a polypeptide of thepresent invention would consist of about 120 amino acids, with acalculated molecular weight of approximately 14,000 daltons(unglycosylated). There appear to be three potential N-glycosylationsites (Asn-Xaa-Ser/Thr) (Neuberger, A. et al., Glycoproteins 5, 450-490,Elsevier Publishing Co., U.S.A. 1972!) at positions 61-63, 91-93, and117-119, which could create a much higher and variable molecular weightdepending upon the expression host, expression conditions, etc.

FIG. 1B shows another embodiment of the present invention, a human cDNAthat contains 153 amino acid residues. Because this lymphokine also is anaturally secreted protein, a hydrophobic leader sequence would beexpected to precede the sequence for the mature secreted form of theprotein. Analysis of the hydrophobicity of the polypeptide andcomparison with a proposed consensus sequence for the processing ofsignal peptides (Periman, D. and Halverson, H., J. Mol. Biol.167:391-409 1983! suggest that cleavage of the precursor polypeptidecould occur following the glycine residue at position 24. Thus, themature polypeptide would be about 129 amino acid residues in length andbegin with a histidine residue. The deduced Mr of the mature secretedprotein is about 15,000 daltons. This molecular weight does not takeinto account potential posttranslational glycosylation of thepolypeptide, which is predicted by the presence of two potentialN-glycosylation sequences (Asn-Xaa-Thr, or Asn-Xaa-Ser, at positions62-64, and 129-131, respectively). Two of these positions appearnaturally conserved between mice and humans.

A variety of methods may be used to prepare the cDNAs of the presentinvention. By way of example, total mRNA is extracted (e.g., as reportedby Berger, S. et al., Biochemistry 18:5143:5149 1979!) from cells (e.g.,a nontransformed human T-cell source) producing polypeptides exhibitingthe desired activity. The double-stranded cDNAs from this total mRNA canbe constructed by using primer-initiated reverse transcription (Verme,I., Blochem. Biophys. Acta, 473: 1-38 1977!) to make first thecomplement of each mRNA sequence, and then by priming for second strandsynthesis (Land, H. et al., Nucleic Acids Res., 9: 2251-2266 1981!).Subsequently, the cDNAs can be cloned by joining them to suitableplasmid or bacteriophage vectors (Rougeon, F. et al., Nucleic AcidsRes., 2, 2365-2378 1975!) or Scherer, G. et al., Dev. Biol. 86, 438-4471981!) through complementary homopolymeric tails (Efstratiadis, A. etal., Cell, 10, 571-585 1977!) or cohesive ends created with linkersegments containing appropriate restriction sites (Seeburg, P. et al.,Nature, 270, 486-495 1977! or Shine, J. et al., Nature, 270, 494-4991977!), and then transforming a suitable host. (See generally,Efstratiadis, A., and Villa-Kormaroff, L., "Cloning of double strandedcDNA" in Setlow, J. and Hollaender, A. (eds.) Genetic Engineering, Vol.1, Plenum Publishing Corp., New York, U.S.A. 1982!.)

A preferred method of obtaining the full-length cloned cDNAs of thisinvention is the procedure developed by H. Okayama and P. Berg (Mol. andCell. Biol., 2: 161-170 1982!). This method has the advantage of placingthe cDNA inserts in a bacterial cloning vector at a position whereby thecDNA can also be directly translated and processed in mammalian cells.Briefly, the first cDNA strand is primed by polydeoxythymidylic acidcovalently joined to one end of a linear plasmid vector DNA. The plasmidvector is then cyclized with a linker DNA segment that bridges one endof the plasmid to the 5' end of the cDNA coding sequence. By employing aDNA fragment containing the Simian Virus 40 (SV40) early region promoterand a linker containing a modified SV40 late region intron, the cDNA canbe expressed in vitro in COS mouse cells without further modification.(See generally, Okayama, H. and Berg, P., Mol. and Cell. Biol.,3:280-289 1983! and Jolly, D. et al., Proc. Nat. Acad. Sci. U.S.A., 80:477-481 1983!, both of which are incorporated herein by reference.)

Once the cDNA library in the Okayama/Berg plasmid vector has beencompleted, the cDNA clones are collected, and random pools can bechecked for the presence of the desired cDNAs .by hybrid selection,translation, and assay (e.g., by measuring synergistic mast cell, or Bcell or T-cell stimulatory activity in cell cultures, the existence ofantigenic determinants, or other biological activities). Pools positiveby these criteria can then be probed with an appropriate subtractedprobe, e.g., cDNA from a B cell line and/or uninduced T-cell line.Thereafter, the positive, probed pools are divided into individualclones which are tested by transfection into a suitable host (such as amammalian cell culture), and the host supernatant assayed for thedesired activity. Positive clones are then sequenced.

The desired cDNA clones can also be detected and isolated byhybridization screening with appropriate mRNA samples (Heindell, H. etal., Cell, 15: 43-54 1978!). Alternatively, the cDNA libraries can bescreened by hybrid selection (Harpold, M. et al., Nucleic Acid Res., 5:2039:2053 1978! or Parnes, J. et al., Proc. Nat. Acad. Sci. U.S.A.,78:2253:2257 1981!) or in Xenopus oocytes (Gurdon, J., Nature, 233:177-182 1971!). (See generally, Villa-Kormaroff, L. et al., Proc. Nat.Acad. Sci. U.S.A., 75:3727-3731 1978!.)

Once identified and obtained in substantially pure form, the cDNA's ofthe present invention can be inserted in their entirety or as fragmentsinto any of a number of cloning and expression vehicles. Additionalmanipulations to create derived nucleotide sequence can includenucleotide base changes, additions and/or deletions to either reflectallelic variations within a species or otherwise modify the DNAsequences as described to allow for production, for example, of otherpolypeptides exhibiting one or more properties (e.g., cellproliferation, immunogenicity, etc.) of the native gene products. By wayof example, those nucleotide sequences derived from the nucleotidesequences disclosed herein in accordance with the techniques well knownto those skilled in the art, such as by chemical synthesis, come withinthe scope of the present invention.

Utilizing these general techniques, a number of lymphokine genes and/orthe purification of proteins encoded by the genes, have been cloned bytransfection into COS cells. Supernatants from transfected COS cellsutilized in experiments described herein include for example: COS-IL-3(Yokota, T. et al., Proc. Natl. Acad. Sci. U.S.A., 81:1070-1074) andCOS-IL-2 (Yokota, T. et al., Proc. Natl. Acad. Sci. U.S.A., 82:68-72(1985)). Co-pending applications relating to the cloning of variouslymphokine genes include U.S. Ser. No. 539,050, filed Oct. 4, 1983; U.S.Ser. No. 590,867, filed Mar. 19, 1984; U.S. Ser. No. 658,183, filed Oct.5, 1984; and U.S. Ser. No. 673,989, filed Nov. 20, 1984 and U.S. Ser.No. 799,699, filed Nov. 19, 1985. All of these publications andapplications are incorporated herein by reference.

In further describing the procedures relating to preparing cDNA clonesof the invention, the cellular source will be considered first, followedby general descriptions of the procedures for isolating mRNA coding fora protein exhibiting the activities of proteins of the presentinvention; the construction of a cDNA library containing the cDNAsequences; isolation of full-length cDNA clones in a plasmid vector andsubsequent expression in mammalian cells; subcloning and expression inbacteria and yeast; and purification and formulation. A more detaileddescription of the entire experimental process will follow thereafter.

Cellular Sources

A preferred source of mRNA encoding the desired polypeptides are cellswhose supernatants contain the B-cell, T-cell and mast cell stimulatingactivities, or other activites associated with the polypeptides of thepresent invention. One such line is the mouse T-cell line Cl.Lyl⁺ 2⁻ /9(A.T.C.C. Accession No. CRL8179) (Nabel, G. et al., Nature, 291:332-334(1981)). In general, suitable T-cells can be obtained from a variety ofsources, such as mammalian (e.g. human) spleen, tonsils and peripheralblood. T-Cell clones, such as those isolated from peripheral bloodT-lymphocytes, may also be used (see, Research Monographs in Immunology,eds. yon Doehmer, H. and Haaf, V.; Section D: "Human T-Cell Clones",vol.8, pgs. 243-333; Elsevier Science Publishers, New York 1985!).

Isolation of mRNA and Construction of a cDNA Library

Total cellular mRNA can be isolated by a variety of well-known methods(e.g., Przybla, A. et al., J. Biol. Chem. 254: 2154-2158 1979!), but thepreferred method is the guanidinium-thiocyanate extraction procedure ofChirgwin et al- (Biochemistry, 18: 5294-5299 1979!). If this method isused, approximately 10 μg of polyA⁺ mRNA, selected on columns of oligo(dT) cellulose, is obtained from 1-2×10⁸ activated T-cells.

The cDNA library from the polyA⁺ mRNA can best be constructed using thepcDV1 vector-primer and the pL1 linker fragment available from the P-LBiochemicals Inc., Milwaukee, Wis.! according to procedures which resultin greatly enriched full-length copies of mRNA transcripts (e.g.Okayama, H. and Berg, P., Mol. Cell Biol., 2, 161-170 1982! and Mol.Cell Biol., 3, 280-289 1983!). The plasmid vector, which contains SV40early promoter and SV40 RNA processing signals, is designed to promoteexpression of the cloned cDNA segment in mammalian cells. If desired,the pcDV1 vector can be modified to contain an NsiI site at the previouslocation of the KpnI site (Yokota, T. et al., Proc. Natl. Acad. Sci.U.S.A., 82:68-72 (1985), which is incorporated herein by reference).

Using the Okayama and Berg procedure, the cyclized vector-cDNApreparation is transformed into a competent bacterial cell, such as E.coli MCl061 cells (Casadaban, M. and Cohen, S., J. Mol. Biol., 138:179-207 1980!) using calcium chloride (Cohen, S. et al., Proc. Nat.Acad. Sci. U.S.A., 69:2110-2114 1972!). Starting with 5 μg of polyA⁺ RNAfrom ConA-stimulated T-cells, about 1×10⁵ independent transformants canbe obtained. Usually, about 104 clones are picked up individually andinoculated into wells of microtiter plates (Flow Laboratories Inc.,McLean, Virginia) containing 200 μl of L-broth, 50 μg/ml of ampicillin,and 7% DMSO. If desired, sublibraries based on the size of cDNA insertare prepared from total cDNA library as described by Okayama, H. Berg,P. (Mol. Cell Biol., 3, 280-299 1983!). Briefly, plasmid DNA is digestedwith SalI, ClaI, and HindIII separately, and electrophoresed in a 1%agarose gel. After staining with ethidium bromide, the gel is slicedinto 7 sections corresponding to cDNA insert sizes of 0 to 1, 1 to 2, 2to 3, 3 to 4, 4 to 5, 5 to 6, and more than 6 kilobases (kb). DNA isextracted from each slice, recyclized with T4 DNA ligase, and used totransform MC1061. All nucleotide sequencing can be performed accordingto the procedure of Maxam, A. and Gilbert, W. (Methods Enzymol.,65:499-560 1980!and Rubin, C. and Schnid, C., Nucl. Acids, Res.,8:4613-4619 1980!), or a dideoxy chain termination protocol (Sanger, F.et al., Proc. Natl. Acad. Sci. U.S.A., 74:5463-5467 1977! withsupercoiled DNA templates.

DNA Transfections into Monkey Cells:

Approximately 1×10⁶ COS cells are seeded onto 60 mmplates the day priorto transfection. Transfections are best performed with 15 μg of plasmidDNA in 1.5 ml of DME containing 50 mM Tris.HCl, pH 7.4, and 400 μg/mlDEAE-Dextran (Pharmacia Fine Chemicals, Uppsala, Sweden). This solutionis then removed after 4 hr and replaced with 2.0 ml DME+4% fetal calfserum. The medium is collected after 72 hr and assayed for the desiredactivities as described above. DNA transfections may be carried out inL-cells and a variety of other cell sources as well (see below).

Cellular Assays

Any of the traditional cellular assays may be utilized in accordancewith established protocols well known to those skilled in the art.T-cell growth factor activity can be determined using factor-dependentT-cell lines; or peripheral blood blasts; B-cells growth factor activitycan be determined using anti-IgM treated B-cells; in mice mast cellgrowth factor activity can be determined using mast cell lines; andcolony stimulating factor activity can be determined using hematopoieticprogeniter cells from bone marrow (or human cord blood) cultured, forexample, in semi-solid medium. Proliferation can be determined either byincorporation of ³ H!thymidine or by a colorimetric assay as describedin Rennick, D. M. et al. J. Immunol. 134:910-914 1985!; andrespectively, Mosmann, T., J. Immunol. Methods 65:55-63 1983!, both ofwhich are incorporated herein by reference. The induction of Ia antigenon B cells can be performed as described in Roehm, N. W. et al., J. Exp.Med. 160:679-694 (1984). Briefly, the Ia positive phenotype isdetermined by staining with anti-I-A^(bd) (D3.137.5.7.) or MK-d6(anti-I-A^(d)) monoclonal antibodies and appropriatefluorescein-conjugated second antibodies. The analysis of stained cellsis accomplished using a fluorescence activated cell sorter. The effectof test samples on the secretion of IgE and IgG₁ by cultures of LPSstimulated B cells is usually determined using isotype specific enzymelinked immunosorbant assays.

Isolation of Related Genes

cDNA clones of the present invention can be used to identify and isolatenucleic acid sequences encoding related genes, such as from othermammals. Because of the frequent low degree of homology betweenhomologous genes, the stringency of hybridization conditions must beadjusted to allow for cross-hybridization between sequences which may beonly 70-80% homologous, or less.

Several different experimental protocols may be used to locate relatedgenes. For example, the human Ck immunoglobulin light chain gene hasbeen isolated using the corresponding mouse Ck gene as a probe (Heiter,P. et al., Cell 22:197-207 1981!) and mouse transplantation antigengenes have been isolated by hybridization to DNA clones encoding theirhuman counterparts (Steinnetz, T. et al., Cell 24:125-134 1981!). Bothreferences are incorporated herein by reference.

For genomic DNA, a preferred method entails plating phage clones from aDNA library containing the homologous genes (Maniatis, T. et al.,"Molecular Cloning, A Laboratory Manual", U.S.A. 1982!) at a density of2×10⁴ to 5×10⁴ plaques per 150 mm plate in an appropriate host strain,such as E. coli LE392. Ten to twenty plates are generally sufficient.

After 10-12 hours' incubation at 37° C., the plates are refrigerated fortwo hours and then a 132 mm nitrocellulose filter is applied to the agarsurface of each plate. The filter is allowed to remain in contact withthe plate for at least five minutes, during which time the filters arekeyed to the plates by puncturing with an ink-filled 22-gauge needle.The filters are then peeled from the plates and incubated successivelyfor at least two minutes first in 250 ml of 0.1N NaOH, 0.5M NaCl; thenin 250 ml of 0.5M Tris.HCl pH 7.5, 1.5M NaCl. The filters are dried onpaper towels and then baked at 80° C. for 4-8 hours.

For hybridization, the filters are wetted in 1× SET (0.15M NaCl, 30 mMTris.HCl pH 8.0, 1 mM Na₂ EDTA), then incubated in a solution of 3× SET,5× Denhardt's (Denhardt, D. T., B.B.R.C. 23:641-646 1966!), 10% dextransulfate, 0.1% sodium dodecyl sulfate (SDS), and 50 μg/ml each poly (rA),poly (rC), and poly (rG), at 65° C. for 2 hrs (1.5-2 ml/filter) withconstant agitation. This solution is then discarded, and the filters arehybridized with a nick-translated probe made from cDNA of the presentinvention (1×10⁸ cpm) in the same solution (fresh), 1.5-2 ml/filter at65° C. for 1 hour and then at 55° C. for 12-20 hours. The filters arethen washed successively in 3× SET, 1× Denhardt's; 0.1% SDS; and 1× SET,0.1% SDS (10-15 ml/filter) at 55° C. for one hour with gentle agitation.The filters are dried on paper towels, then autoradiographed for 12-24hours with appropriate film and an intensifying screen. Hybridizingplaques are picked from agar plates with sterile Pasteur pipets, andeach is expelled into 1 ml of 0.1M NaCl, 0.01M Tris.HCl pH 7.5, 10 mmMgCl₂, 100 μg/ml gelatin, with 50 μl of CHCl₃ added. After at least 4-8hours in the cold, the phage from each plaque are rescreened at lowdensity (2000-4000 plaques/150 mm plate) by a procedure identical tothat described above.

Expression in E. Coli, in Yeast and in Cell Culture

Prokaryotes, such as E. coli, are very suitable for expression of thepolypeptides of the present invention (see, for example, U.S. Pat. Nos.4,338,397 and 4,411,994), provided glycosylation is not desired.

To obtain high expression levels, promoters should be utilized, such asthe β-lactamase (penicillinase) and lactose promoter systems (Chang etal., Nature, 275:615 1978!; Itakura et al., Science, 198: 1056 1977!;Goeddel et al., Nature 281:544 1979! or a tryptophan (trp) promotersystem (Goeddel et al., Nucleic Acids Res., 8:4057 1980!) in conjunctionwith ribosome binding sequences.

Those skilled in the art will realize that not only prokaryotes but alsoeukaryotic microbes, such as yeast, may also be used in proteinproduction. Saccharomyces cerevisiae is a preferred eukaryoticmicroorganism. Suitable promoting sequences in yeast vectors include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem., 255:12073-12080 1980!) or other glycolytic enzymes (Hess et al.,Adv. Enzyme Reg., 7:149-167 1969!; Holland et al., Biochemistry,17:4900-4907 1978!). Other promoters that have the additional advantageof transcription controlled by growth conditions may be used. Basicallyany plasmid vector containing a yeast-compatible promoter, an origin ofreplication and termination sequences is suitable.

A preferred method of making polypeptides employing the cDNA's of thepresent invention utilizes the yeast mating pheromone α-factor secretorypathways (Julius, D. et al., Cell 32:839-852 1983!). S. cerevisiaesecretes mating-type specific oligopeptide pheromones. MATα cellssecrete α-factor, which induces the growth arrest of MATαcells at Glphase of the cell cycle (Thorner, J., The Molecular Biology of the YeastSaccharomyces, Cold Spring Harbor Laboratory, New York 1981!; seeparticularly pages 153-180). The α-factor is initially synthesized as alarger precursor molecule consisting of an NH₂ -terminal signal sequenceof about 20 amino acids, followed by an additional 60 amino acid leadersequence and ending with four identical tandem repeats of the matureα-factor sequence. The repeats are separated from each other by six oreight amino acid spacers (Lys-Arg-Glu-Ala-Glu-Ala andLys-Arg-Glu-Ala-Glu- or Asp-!-Ala-Glu-Ala). This prepro-α-factor iscleaved at several specific sites. The first processing is the cleavageof the COOH-terminal side of the Lys-Arg pair of the spacer sequencecatalysed by the KEX2 product (Julius et al., Cell 37:1075-1089 1984!).A carboxypeptidase-B like enzyme cleaves at the NH₂ -terminal side ofthe Lys-Arg pair. The final step is the removal of Glu-Ala or Asp-Alapairs by diaminopeptidease, which is encoded by the STE13. Brake, J. etal., (Proc. Nat. ACad. Sci. U.S.A. 81:4642-4646 1984!) have shown thatthe fusion of the sequence encoding mature human proteins to the firstprocessing site allowed secretion of such proteins.

A 1.7 kb EcoRI fragment carrying the MFαl gene (Kurjan, J. andHershowitz, I., Cell. 30:933-943 1982!) is cloned into the EcoRIrestriction site of M13mp8 (Viera, J. and Messing, J., Gene 19:259-2681982!). In order to introduce a HindIII site after the lysine codon ofthe first spacer region, the synthetic oligonucleotideTCTTTTATCCAAAGATACCC is hybridized to the single strand M13-Mfα1 DNA andthe oligonucleotide primer extended by DNA polymerase I Klenow fragment.After Sl nuclease treatment, the DNA is cleaved with EcoRI and thefragment carrying the MFαl promoter and leader sequence cloned into theEcoRI and filled-in HindIII restriction sites of PUC8 (Viera, J. andMessing, J. above). One plasmid with the desired structure can beisolated (designated PMFα4Δl ). The pMFαΔl is cleaved with HindIII andpartially filled in with DNA polymerase I Klenow fragment in thepresence of dATP and DGPT. The DNA is treated with mung bean nuclease,and the oligonucleotide linker GCCTCGAGGC attached. The resultantplasmid (designated PMFα5) will have a StuI cleavage site immediatelyafter the arginine codon, followed by the XhoI restriction site. An S.cerevisiae-E. coli shuttle vector (pTRP584) can be constructed asfollows: the PstI-XbaI fragment carrying 2 μm plasmid replication origin(Broach, J. above) is cloned into the ClaI restriction site of PTRP56(Miyajima et al., Mol. Cell. Biol. 4: 407-414 1984!) and the StuIrestriction site within the TRPl-ARSl fragment converted into a PvuIIrestriction site by PvuII linker insertion. The KpnI restriction site inthe original pTRP56 is converted to XhoI by the XhoI linker insertion.The general secretion vector pMFα8 is then obtained by insertion of theBglII-XhoI fragment of pMFα5 into the BamHl-XhoI restriction sites ofpTRP584.

Those skilled in the art will realize that cDNA clones of the presentinvention may then be readily inserted into the pMFα8 vector andsubsequently transformed in yeast for production of the desiredpolypeptides. By way of example, a BamHI fragment carrying an entirecDNA of the present invention is cloned into the BamHI restriction siteof M13mp8 (Viera, J. and Messing, J., Gene 19:259-268 1982!). In orderto make a double stranded fragment carrying the mature protein codingsequence, a complementary oligonucleotide primer is constructed. Thisprimer is then hybridized to a single stranded M13mp8 vector carryingcDNA of the present invention, and is extended by DNA polymerase IKlenow fragment. The double stranded DNA is cleaved with BamHI and thenthe single strand region is removed by mung bean nuclease. The doublestranded fragment carrying the mature protein coding sequence of thepresent invention is then isolated and cloned into the StuI restrictionsite of the general secretion vector pMFχ8. This plasmid DNA (carryingthe TRP1 gene) can be introduced into yeast cells by the lithium acetatemethod (Ito, H. et al., J. Bacteriol. 153:163-168 1983!) andtransformants selected in synthetic medium lacking tryptophan.Transformants are then grown in a common medium supplemented with 0.5%casamino acids. To harvest the yeast cells, they are first resuspendedin phosphate-buffered-saline (PBS) containing 1 mM PMSF and thendisintegrated by vigorous shaking with acid washed glass beads. Clearsupernatant is obtained by centrifugation at 10,000 rpm for 15 min.

In addition to microorganisms, cell cultures derived from multicellularorganisms (especially mammalian cells) may also be used as hosts.Examples of such useful host cell lines are HeLa cells, L cells, Chinesehamster ovary ceil lines, and baby hamster kidney cell lines. Expressionvectors for such cells ordinarily include, as necessary, an origin ofreplication, a promoter located in front of the gene to be expressed,along with any required ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences. Whenused in mammalian cells, the expression vector often has controlfunctions provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, and most frequentlySV-40. (See, e.g., U.S. Pat. No. 4,399,216 and Ghuysen, D. and Fiers,W., J. of Mol. and Appl. Genetics 1:385-394 1982!.)

Purification and Formulations

The polypeptides of the present invention expressed in E. coli, in yeastor in other cells can be purified according to standard procedures ofthe art, including ammonium sulfate precipitation, fractionation columnchromatography (e.g., ion exchange, gel filtration, electrophoresis,affinity chromatography, etc.) and ultimately crystallization (seegenerally "Enzyme Purification and Related Techniques," Methods inEnzymology, 22:233-577 1977! and Scopes, R., Protein Purification:Principles and Practice, Springer-Verlag, New York 1982!). Oncepurified, partially or to homogeneity, the polypeptides of the inventionmay be used for research purposes, e.g., as a supplement to cell growthmedia (e.g., minimum essential medium Eagle, Iscove's modified DulbeccoMedium or RPMI 1640; available from Sigma Chemical Company, St Louis,Mo. and GIBCO Division, Chagrin Falls, Ohio) and as an antigenicsubstance for eliciting specific immunoglobulins useful in immunoassays,immunofluorescent stainings, etc. (See generally, Immunological Methods,Vols. I & II, Eds. Lefkovits, I. and Perhis, B., Academic Press, NewYork, N.Y. 1979 & 1981!; and Handbook of Experimental Immunology, ed.Weir, D., Blackwell Scientific Publications, St. Louis, Mo. 1978!.)

The polypeptides of the present invention may also be used inpharmaceutical compositions, e.g., to enhance natural defense againstvarious infections. Thus, patients with rheumatoid arthritis, in need ofa transplant, or with immunodeficiency caused by cancer chemotherapy,advanced age, immunosuppressive agents, etc., may be treated with suchpolypeptides. The compositions can selectively stimulate variouscomponents of the immune system, either alone or with other agents wellknown to those skilled in the art. In particular, the compositions mayinclude other immune-reactive agents, such as lymphokines (e.g. IL-1,IL-2, etc.), any of the cloning stimulating factors, immunoglobulins,etc., in view of the demonstrated synergistic activities of thepolypeptides of the present invention. The polypeptides will also finduse in situations (in vivo or in vitro) in which enhanced cellularproliferation or immunoglobulin production is desired.

For preparing pharmaceutical compositions containing the polypeptidesdescribed by this invention, these polypeptides are compounded byadmixture with preferably inert, pharmaceutically acceptable carriers.Suitable carriers and processes for their preparation are well known inthe art (see, e.g., Remington's Pharmaceutical Sciences and U.S.Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa.1984!). The preferred course of administration is parenteral and caninclude use of mechanical delivery systems.

Preferably, the pharmaceutical composition is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The quantity of activecompound in a unit dose of preparation may be varied or adjusted from 1μg to 100 mg, according to the particular application and the potency ofthe active ingredient. The composition can, if desired, also containother therapeutic agents.

The dosages may be varied depending upon the requirement of the patient,the severity of the condition being treated and the particular compoundbeing employed. Determination of the proper dosage for a particularsituation is within the skill of the art. Generally, treatment isinitiated with smaller dosages which are less than the optimum dose ofthe compound. Thereafter, the dosage is increased by small incrementsuntil the optimum effect under the circumstances is reached. Forconvenience, the total daily dosage may be divided and administered inportions during the day.

The following experimental information and data are offered by way ofexample and not by way of limitation.

EXPERIMENTAL EXAMPLE I

This example demonstrates the isolation of a cDNA clone encodingpolypeptides of the present invention that are active, in particular, onmouse cells.

A. Cloned Helper T Cells

1) A clone of T-cells Cl.Lyl⁺ 2⁻ /9 (A.T.C.C. Accession No. CRL8179)expressing the Thy 1⁺ Ly 1⁺ 2⁻ phenotype is continuously maintained at0.5×10⁻⁵ cells/ml in Dulbecco's Modified Eagles medium (DME) with 10%heat-activated fetal calf serum, 5×10⁻⁵ M 2-mercaptoethanol (2-ME), 2 mMglutamine, non-essential amino acids, and essential vitamins conditionedwith 25% supernatants from Concanavalin A (Con A)-activated mouse Balb/cspleen cells.

2) Con A-activation of Cl.Lyl⁺ 2⁻ /9 cells. The cells are cultured at5×10⁵ /ml in DME with 4% heat-inactivated fetal calf serum, 5×10⁻⁵ M2-ME, 2 mM glutamine, non-essential amino acids, essential vitamins and2 μg/ml Con A. After 12-14 hrs. incubation at 37° C. in 10% CO₂, thecell suspension is centrifuged at 1500 rpm for 10 minutes. The cellpellets are collected and frozen immediately at -70° C. The supernatantsare filtered (Nalgene-0.22 microns) and stored at -20° C. as a source ofgrowth factors. Aliquots of the supernatant are assayed for MCGFactivity (see below) to verify the induction of the line by the Con Atreatment.

B. Cellular Assays

1) TCGF Assay: T-cell growth factor activity was determined by ³H!thymidine incorporation with the use of HT-2 T-cells as described inNabel, G. et al., Proc. Natl. Acad. Sci. U.S.A., 78:1157 (1981), whichis incorporated herein by reference. HT-2 cells were cultured at 5×10³cells/well (Falcon microtiter trays) in modified DME, 4% FCS, and variedconcentrations of test supernatants. Then 0.5 μCi ³ H!thymidine wasadded to each culture for the last 4 hr of a 24-hr incubation period.The cells were then harvested onto glass fiber filters, andradioactivity was measured in a liquid scintillation spectrometer.

2) MCGF Assay: MCGF activity was determined by a colorimetric assay(Mosmann, T. supra) with the use of MC/9 mast cells (A.T.C.C. AccessionNo. CRL8306). Briefly, MC/9 cells were cultured in flat-bottom Falconmicrotiter trays (10⁴ cells/well) in DME supplemented with 4% FCS, 50 μM2-mercaptoethanol, 2 mM glutamine, non-essential amino acids, essentialvitamins, and varied concentrations of test supernatants in a finalvolume of 0.1 ml. Fifty micrograms of 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma) in 10μl of phosphate-buffered saline were added to each cell culture after a20-hr incubation. Four hours later, 0.1 ml of 0.04M HCl in isopropanolwas added to solubilize the colored reaction product. The absorbance at570 nm (reference 630 nm) was measured on a Dynatek MicroelisaAutoreader (MR580).

3) Ia Antigen Assay: Several DBA/2 mice (2-3 months old) were sacrificedand the spleens obtained surgically. The erythrocytes were lysed byhypotonic shock using 0.87% ammonium chloride. Then the T-cells werelysed by using cytotoxic monoclonal antibodies directed againstT-cell-specific surface markers (Thy-1, Lyt-1 and Lyt-2) followed byincubation in rabbit complement. The dead cells were then removed usingficoll-hypaque density gradients. Adherent cells had been removedpreviously by adherence to plastic petri dishes at 37° C. At this timethe cells were washed, counted and scored for viability. One millioncells were incubated in 0.5 ml of tissue culture medium (RPMI 1640 orMinimal essential medium-MEM/Earle's salts) (Gibco) supplemented with10% fetal calf serum, 2-mercaptoethanol and various antibiotics(penicillin, streptomycin and gentamicin). In experiments where thepositive control consisted of supernatants from T-cells induced with theT-cell mitogen Concanavalin A, 10 mg/ml (final concentration) ofalpha-methyl mannoside was added to neutralize the mitogen. After 24hours incubation, the cells were harvested and prepared for stainingwith anti-I-A^(d) monoclonal antibodies (in some experiments MK-D6(I-A_(d)) and in some other experiments the monoclonal antibodyD3.137.5.7 (anti-I-Abd) were used). These antibodies were used as firststage antibodies conjugated to either the hapten N.I.P. (MK-D6) orbiotin (D3.137.5.7). The staining was then completed by incubating thecells with fluoresceinated second-stage reagents (either anti-NIPantibodies or avidin). The intensity of fluorescence staining was thendetermined using either a fluorescence-activated cell sorter(Becton-Dickinson, Mountain View, Calif.) or a Cytofluorograph (OrthoDiagnostics, cambridge, Mass.).

4) Measurement of IgE and IgG₁ Enhancing Activity: T cell-depletedspleen cell suspensions were prepared by forcing spleen fragmentsthrough a 200-mesh wire screen. The cell suspensions were washed andincubated for 15 minutes on ice with anti-Thy-1.2. The cells were thenpelleted and resuspended in rabbit complement (Lo-Tox M, CedarlaneLaboratories, Hornby, Ont., Canada) diluted in RPMI-1640 containing 25mM Hepes, pH 7.2, and 0.3% bovine serum albumin. Cells were incubatedwith complement for 45 minutes at 37° and the dead cells were removed bycentrifugation over Ficoll (density 1.119 g/ml, Sigma, St. Louis, Mo.).All steps except the complement treatment were performed in Hanksbalanced salts solution plus 0.3% bovine serum albumin. B cells werepurified by incubating spleen cell suspensions with a mixture ofmonoclonal antibodies to Thy-1, L₃ T₄, Lyt-2, MAC-1, and the antibodyRB6-8C5, followed by complement treatment and final centrifugation, asdescribed above, for the removal of dead cells. The cells were thenstained with fluorescein-conjugated RA3-6B2 and the cells separated intolarge and small positive fractions with a FACS-IV fluorescence-activatedcell sorter (Becton-Dickinson, Mountain View, Calif.). Cells werecultured in round-bottom 96-well plates (Flow Laboratories, McLean, Va.)in RPMI-1640 medium (Gibco) plus penicillin, streptomycin, glutamine,2-mercaptoethanol (5×10⁻⁵ M) and 10% fetal calf serum (Hyclone, Logan,Utah). The final concentrations of cells were: B cells and T-depletedspleen cells, 5×10⁵ /ml; spleen cells, 1×10⁶ cells/ml. Cells werecultured at twice the final cell concentration with 4 μg/ml ofSalmonella typhimurium LPS (Sigma). One day later Cl.Lyl⁺ 2⁻ /9supernatant and/or IFN-γ were added in 0.1 ml of medium. Culturesupernatants were harvested 7 days after initiation of the cultures andfrozen until assayed. Balb/cByJ mice were used in all experiments exceptwhere noted. For isotype determinations, polyvinyl chloride 96-wellplates (Dynatech, Alexandria, Va.) were coated for 1 hour with theappropriate first step isotype-specific antibody at concentrations of0.5 to 2.0 pg/ml. These plates were blocked with phosphate-bufferedsaline plus 0.1% bovine serum albumin and 0.04% Tween 20. Standard curvesolutions and supernatants to be tested were added in 0.1 ml, alldilutions being made in RPMI-1640 plus 5% fetal calf serum After threehours at room temperature, the plates were washed in phosphate-bufferedsaline plus 0.04% Tween 20 and the appropriate NIP or biotin conjugatedsecond step antibody at concentrations of 0.25 to 2.0 μg/ml. Afterincubation for 1 hour at room temperature, these plates were washed asbefore an an optimum concentration of either horseradish peroxidaseconjugated monoclonal anti-NIP or horseradish peroxidase conjugatedavidin (Vector Laboratories, Burlingame, Calif.) was added. One hourlater, the plates were washed and 0.1 ml was added of a substratesolution containing 1 mg/ml 2,2'-azinobis(3-ethylbenzthiazolinesulfonicacid) (Sigma) and 0.003H₂ O₂ in 0.1M Na₂ HPO₄ and 0.05M citric acid. Thereaction was stopped by the addition of 0.05 ml of 0.2M citric acid andthe plates read on a Dynatech ELISA reader. For the IgE ELISA, plateswere coated with a monoclonal anti-IgE, EM95, and the second step wasNIP-conjugated purified rabbit anti-IgE antibody. IgM, IgG_(2a),IgG_(2b), IgG₃, and IgA ELISA used the same isotype-specific rabbitantibodies as a first step and as a NIP-conjugated second step. IgG₁ wasassayed using unconjugated and biotin-conjugated preparations of thesame IgG₁ -specific rabbit antibodies. Specificity of eachisotype-specific ELISA was tested by assaying 20 μg/ml solutions of twoor more purified monoclonal immunoglobulins of each of the other 6isotypes under the same conditions used for the appropriate isotype. Inall cases, the 20 μg/ml solution gave a reading no higher than thelowest concentration on the standard curve (ranging from 1.25 ng/ml forIgE to 3.1 ng/ml for IgM). Further evidence of specificity can bededuced from the independent variation of isotypes under differentculture conditions.

C. Isolation of mRNA from T Cells

1) Total cellular DNA was isolated from cells using the guanidineisothiocyanate procedure of Chirgwin, J. et al., (Biochemistry, 18:5294-5299 1979!).

Frozen cell pellets from ConA-induced helper cells (12 hrs afterstimulation) were suspended in guanidine isothiocyanate lysis solution.Twenty ml of lysis solution was used for 1.5×10⁸ cells. Pellets wereresuspended by pipetting, then DNA was sheared by 4 passes through asyringe using a 16 gauge needle. The lysate was layered on top of 20 mlof 5.7M CsCl, 10 mM EDTA in 40 ml polyallomer centrifuge tube. Thissolution was centrifuged at 25,000 rpm in a Beckman SW28 rotor (BeckmanInstruments, Inc., Palo Alto, Calif.) for 40 hrs at 15° C. The guanidineisothiocyanate phase containing DNA was pipetted off from the top, downto the interface. The walls of the tube and interface were washed with2-3 ml of guanidine isothiocyanate lysis solution. The tube was cutbelow the interface with scissors, and the CsC1 solution was decanted.RNA pellets were washed twice with cold 70% ethanol. Pellets were thenresuspended in 500 μl of 10 mM Tris.HCl pH 7.4, 1 mM EDTA, 0.05% SDS. 50μl of 3M sodium acetate was added and RNA was precipitated with 1 mlethanol. About 0.3 mg total RNA was collected by centrifuging and thepellets washed once with cold ethanol.

2) PolyA⁺ mRNA isolation:

Washed and dried total RNA pellet was resuspended in 900 μl of oligo(dT) elution buffer (10 mM Tris.HCl, pH 7.4, 1 mMEDTA, 0.5% SDS). RNAwas heated for 3 min. at 68° C. and then chilled on ice. 100 μl of 5MNaCl was added. The RNA sample was loaded onto a 1.0 ml oligo (dT)cellulose column (Type 3, Collaborative Research, Waltham, Mass.)equilibrated with binding buffer (10 mM Tris.HCl pH 7.4, 1 mM EDTA, 0.5MNaCl, 0.5% SDS.). Flow-through from the column was passed over thecolumn twice more. The column was then washed with 20 ml binding buffer.PolyA⁺ mRNA was collected by washing with elution buffer. RNA usuallyeluted in the first 2 ml of elution buffer. RNA was precipitated with0.1 volume 3M sodium acetate (pH 6) and two volumes of ethanol. The RNApellet was collected by centrifugation, washed twice with cold ethanol,and dried. The pellet was then resuspended in water. Aliquots werediluted, and absorbance at 260 nm was determined.

D. cDNA Library Construction:

1) Preparation of vector primer and oligo dG-tailed linker DNAs:

The procedure of Okayama & Berg (Mol. & Cell. Biol. 2:161-170 1982!) wasused with only minor modifications and adapted to the pcDV1 and pLlplasmids described by Okayama & Berg (Mol. & Cell. Biol. 3:380-3891983!). Specifically, a modified pcDV1 plasmid containing an NsiI siteat the previous location of the KpnI site.

An 80 μg sample of pcDV1 DNA was digested at 30° C. with 20 U of KpnIendonuclease in a reaction mixture of 450 μl containing 6 mM Tris.HCl(pH 7.5), 6 mM MgCl₂, 6 mM NaCl, 6 mM 2-ME, and 0.1 mg of bovine serumalbumin (BSA) per ml. After 16 hr the digestion was terminated with 40μl of 0.25 M EDTA (pH 8.0) and 20 μl of 10% sodium dodecyl sulfate(SDS); the DNA was recovered after extraction with water-saturated 1:1phenol-CHCl₃ (hereafter referred to as phenol-CHCl₃) and ethanolprecipitation. Homopolymer tails averaging 60, but not more than 80,deoxythymidylate (dT) residues per end were added to the NsiIendonuclease-generated termini with calf thymus terminal transferase asfollows: The reaction mixture (38 μl ) contained sodium cacodylate-30 mMTris.HCl pH 6.8 as buffer, with 1 mM CoCl₂, 0.1 mM dithiothreitol, 0.25mM dTTP, the NsiI endonuclease-digested DNA, and 68 U of the terminaldeoxynucleotidyl transferase (P-L Biochemicals, Inc., Milwaukee, Wis.).After 30 min. at 37° C. the reaction was stopped with 20 pl of 0.25 MEDTA (pH 8.0) and 10 pl of 10% SDS, and the DNA was recovered afterseveral extractions with phenol-CHCl₃ by ethanol precipitation. The DNAwas then digested with 15 U of EcoRI endonuclease in 50 μl containing 10mM Tris.HCl pH 7.4, 10 mM MgCl₂, 1 mM dithiothreitol, and 0.1 mg of BSAper ml for 5 hr at 37° C. The large fragment, containing the SV40polyadenylation site and the pBR322 origin of replication andampicillin-resistance gene, was purified by agarose (1%) gelelectrophoresis and recovered from the gel by a modification of theglass powder method (Vogelstein, B. & Gillespie, D., Proc. Nat. Acad.Sci. 76:615-619 1979!). The dT-tailed DNA was further purified byabsorption and elution from an oligo (dA)-cellulose column as follows:The DNA was dissolved in 1 ml of 10 mM Tris.HCl pH 7.3 buffer containing1 mMEDTA and 1M NaCl, cooled at 0° C., and applied to an oligo(dA)-cellulose column (0.6 by 2.5 cm) equilibrated with the same bufferat 0° C. and eluted with water at room temperature. The eluted DNA wasprecipitated with ethanol an dissolved in 10 mM Tris.HCl pH 7.3 with 1mM EDTA.

The oligo (dG) tailed linker DNA was prepared by digesting 75 μg of pL1DNA with 20 U of PstI endonuclease in 450 μl containing 6 mM Tris.HCl pH7.4, 6 mM MgCl₂, 6 mM 2-ME, 50 mM NaCl, and 0.01 mg of BSA per ml. After16 hr at 30° C. the reaction mixture was extracted with phenol-CHCl₃ andthe DNA was precipitated with alcohol. Tails of 10 to 15 deoxyguanylate(dG) residues were then added per end with 46 U of terminaldeoxynucleotidyl transferase in the same reaction mixture (38 μl) asdescribed above, except that 0.1 mM dGTP replaced dTTP. After 20 min. at37° C. the mixture was extracted with phenol-CHCl₃, and after the DNAwas precipitated with ethanol it was digested with 35 U of HindIIIendonuclease in 50 μl containing 20 mM Tris.HCl pH 7.4, 7 mM MgCl₂, 60mM NaCl, and 0.1 mg of BSA at 37° ° C. for 4 hr. The small oligo(dG)-tailed linker DNA was purified by agarose gel (1.8%)electrophoresis and recovered as described above.

2) cDNA Library Preparation:

Step 1: cDNA synthesis. The reaction mixture (10 μl) contained 50 mMTris.HCl pH 8.3, 8 mM MgCl₂, 30 mM KCl, 0.3 mM dithiothreitol, 2 mM eachdATP, dTTP, dGTP, and dCTP, 20 μCi ³² p-dCTP (3000 Ci/mmole), 3 μgpolyA⁺ RNA from Con-A induced T-cells, 60 units TNasin (Biotec, Inc.,Madison, Wis.), and 2 μg of the Vector-primer DNA (15 pmol of primerend), and 45 U of reverse transcriptase. The reaction was incubated 60min at 42° C. and then stopped by the addition of 1 μl of 0.25M ETDA (pH8.0) and 0.5 μl of 10% SDS; 40 μl of phenol-CHCl₃ was added, and thesolution was blended vigorously in a Vortex mixer and then centrifuged.After adding 40 μl of 4M ammonium acetate and 160 μl of ethanol to theaqueous phase, the solution was chilled with dry ice for 15 min., warmedto room temperature with gentle shaking to dissolve unreacteddeoxynucleoside triphosphates that had precipitated during chilling, andcentrifuged for 10 min. in an Eppendorf microfuge. The pellet wasdissolved in 10 μl of 10 mM Tris.HCl pH 7.3 and 1 mM EDTA, mixed with 10μl of 4M ammonium acetate, and reprecipitated with 40 μl of ethanol, aprocedure which removes more than 99% of unreacted deoxynucleotidetriphosphates. The pellet was rinsed with ethanol.

Step 2: Oligodeoxycytidylate oligo (dC)! addition. The pellet containingthe plasmid-cDNA:mRNA was dissolved in 20 μl of 140 mM sodiumcacodylate-30 mM Tris.HCl pH 6.8 buffer containing 1 mM CoCl₂, 0.1 mMdithiothreitol, 0.2 μg of poly (A), 70 μM dCTP, 5 μCi ³² p-dCTP, 3000Ci/mmole, and 60 U of terminal deoxynucleotidyl transferase. Thereaction was carried out at 37° C. for 5 min. to permit the addition of10 to 15 residues of dCMP per end and then terminated with 2 μl of 0.25MEDTA (pH 8.0) and 1 μl of 10% SDS. After extraction with 20 μl ofphenol-CHCl₃, the aqueous phase was mixed with 20 μl of 4M ammoniumacetate, the DNA was precipitated and reprecipitated with 80 μl ofethanol, and the final pellet was rinsed with ethanol.

Step 3: HindIII endonuclease digestion. The pellet was dissolved in 30μl of buffer containing 20 mM Tris.HCl pH 7.4, 7 mM MgCl₂, 60 mM NaCl,and 0.1 mg of BSA per ml and then digested with 10 U of HindIIIendonuclease for 2 hr at 37° C. The reaction was terminated with 3 μl of0.25 M EDTA (pH 8.0) and 1.5 μl of 10% SDS and, after extraction withphenol-CHCl₃ followed by the addition of 30 μl of 4M ammonium acetate,the DNA was precipitated with 120 μl of ethanol. The pellet was rinsedwith ethanol and then dissolved in 10 μl of 10 mM Tris.HCl (pH 7.3) and1 mM EDTA, and 3 μl of ethanol was added to prevent freezing duringstorage at -20° C.

Step 4: Cyclization mediated by the oligo (dG)-tailed linker DNA. A 9 μlsample of the HindIII endonuclease-digested oligo (dC)-tailed cDNA;mRNAplasmid (about 90% of the sample) was incubated in a mixture (90 μl)containing 10 mM Tris.HCl pH 7.5, 1 mM EDTA, 0.1M NaCl, and 1.8 pmol ofthe oligo (dG)-tailed linker DNA at 65° C. for 5 min., shifted to 42° C.for 60 min, and then cooled to 0° C. The mixture (90 μl) was adjusted toa volume of 900 μl containing 20 mM

Tris.HCl pH 7.5, 4 mM MgCl₂, 10 mM (NH₄)₂ SO₄, 0.1M KCl, 50 μg of BSAper ml, and 0.1 mM β-NAD; 6 μg of E. coli DNA ligase were added and thesolution was then incubated overnight at 12° C.

Step 5: Replacement of RNA strand by DNA. To replace the RNA strand ofthe insert, the ligation mixture was adjusted to contain 40 μM of eachof the four deoxynucleoside triphosphates, 0.15 mMb-NAD, 4 μg ofadditional E. coli DNA ligase, 16 U of E. coli DNA polymerase I (PolI,)and 9 U of E. coli RNase H. This mixture (960 μl) was incubatedsuccessively at 12 ° C. at room temperature for 1 hr to promote optimalrepair synthesis and nick translation by PolI.

Step 6: Transformation of E. coli. Transformation was carried out usingminor modifications of the procedure described by Cohen et al. (Proc.Nat. Acad. Sci. U.S.A., 69:2110-2114 1972!). E. coli K-12 strain MC1061(Casadaban, M. and Cohen, S., J. Mol. Biol. 138: 179-207 1980!) wasgrown to 0.5 absorbancy unit at 600 nm at 37° C. in 300 ml of L-broth.The cells were collected by centrifugation, suspended in 30 ml of 10 mMPipes. pH 7, 60 mM CaCl₂, 15% glycerol and centrifuged at 0° C. for 5min. The cells were resuspended in 24 ml of the above buffer andincubated again 0° C. for 5 min.; then, 1.0 ml aliquots of the cellsuspensions were mixed with 0.1 ml of the DNA solution (step 5) andincubated at 0° C. for 20 min. Next the cells were kept at 42° C. for 2min. and thereafter at room temperature for 10 min.; then 1 liter ofL-broth was added, and the culture was incubated at 37° C. for 60 min.Ampicillin was added to a concentration of 50 μg/ml. The culture wasshaken for an additional 10 hrs. at 37° C. Dilutions of this culturewere spread on L-broth agar containing 50 μg/ml ampicillin. Afterincubation at 37° C. for 12 to 24 hr, individual colonies were pickedwith sterile tooth-picks. In all, approximately 1×10⁵ independent cDNAclones were generated.

E. Screening of the T-cell cDNA Library by DNA Transfections:

10⁴ single clones were picked at random from the T-cell cDNA library andpropagated individually in wells of microtiter dishes containing 200 plL-broth with ampicillin at 50 μg/ml and dimethyl sulfoxide at 7%. Tofocus only on the novel MCGF activity, 53 IL-3 cDNA clones and oneGM-CSF cDNA clone identified by hybridization with the appropriate ³²P-labelled cDNA probes were eliminated as follows: Each plate of 96cultures was replicated onto nitrocellulose filters for hybridizationscreening. Hybridizations were performed in 6XSSPE (1XSSPE=180 mM NaCl;10 mM sodium phosphate, pH 7.4; 1 mm EDTA), 0.1% SDS, 100 μg/ml E. colitRNA, 50% formamide, for 16 hrs. at 42° C. Hybridizing clones wereidentified by autoradiography of the washed filters. These clones wereremoved by sterilizing the microtiter wells containing these clones withethanol prior to the preparation of clone pools. Pools containing up to48 cDNA clones were prepared from the microtiter cultures. Two hundredsuch pools were grown up in 1 liter cultures of L-broth containing 100μg/ml ampicillin. Plasmid DNA was isolated from each culture andpurified by twice banding through CsCl gradients. The DNA representingeach pool was transfected into COS monkey cells as follows.

One day prior to transfection, approximately 10⁶ COS monkey cells wereseeded onto individual 100 mm plates in DME containing 10% fetal calfserum and 2 mM glutamine. To perform the transfection, the medium wasaspirated from each plate and replaced with 4 ml of DME containing 50 mMTris.HCl pH 7.4, 400 μg/ml DEAE-Dextran and 50 μg of the plasmid DNAs tobe tested. The plates were incubated for four hours at 37° C., then theDNA-containing medium was removed, and the plates were washed twice with5 ml of serum-free DME. DME containing 150 μM chloroquine was added backto the plates which were then incubated for an additional 3 hrs at 37°C. The plates were washed once with DME and then DME containing 4% fetalcalf serum, 2 mM glutamine, penicillin and streptomycin was added. Thecells were then incubated for 72hrs at 37° C. The growth medium wascollected and evaluated in the various bioassays.

An initial set of plasmid pools was screened primarily by usingproliferation assays with the HT-2 and MC/9 cell lines. Among the first110 pools assayed on these two cell lines, eight produced significantactivity in the HT-2 assay. Several of these pools had weak butsignificant MCGF activity, but because the MCGF activities weregenerally weaker and more variable, we did not rely on this assay foridentifying positive pools.

Approximately half of the COS supernatants from the random pooltransfections were also assayed for Ia inducing activity on mouse Bcells. Among the pools tested, each pool shown to be active for TCGFactivity was found also to have Ia inducing activity. Thus, there was aperfect correlation between the TCGF activity and the Ia inducingactivity.

F. Isolation of Functional Mouse cDNA Clones that Express TCGF and MCGFActivities

One pool, 2A, which was reproducibly the most active in all assays, wassubdivided into smaller subpools representing horizontal and verticalrows of the 48 well microtiter plate. One horizontal and one verticalsubpool were positive for both MCGF and TCGF activities. The singleclone, 2A-E3, common to both subpools was then grown individually andits plasmid DNA was transfected as before. The resulting COS supernatantwas then assayed for the presence of various activities, includingcolony formation, MCGF, TCGF, Ia inducing, and IgE and IgG, enhancingactivities.

A 366 base-pair-long PstI fragment isolated from clone 2A-E3 (FIG. 1A)and labelled with ³² P was used as a probe to screen pools which hadbeen positive for biological activity as well as other untested pools.The screening was performed by hybridization to filters replicated withthe microtiter cultures as described above. Nine hybridizing clones wereisolated and their DNA analyzed by restriction mapping. All pools whichexhibited biological activity contained at least one hybridizing clonewhich shared a common restriction cleavage map with clone 2A-E3. Thefrequency of hybridizing clones among the 10⁴ which were picked suggestsa frequency of approximately 0.2% in the total library. Of thehybridizing clones which were tested, approximately 90% expressed afunctional protein.

G. Some Biological Activities of Clone 2A-E3

When supernatant from COS cells transfected with the single 2A-E3 cloneis tested for TCGF activity on HT-2 cells, the dose response curvereaches the same maximum level as seen with supernatant from Cl.Lyl⁺ 2⁻/9 cells (FIG. 3A). Even at saturating levels, however, the COSsupernatant does not achieve the same level of stimulation obtained withrecombinant IL-2. When the same COS supernatant is tested on MC/9 mastcells, the maximal stimulation is approximately the same as withrecombinant IL-3 (FIG. 3B). These results, employing a colorimetricassay (Mosmann, T., supra), were also confirmed using incorporation of ³H!thymidine.

The COS-expressed material of clone 2A-E3 was also tested for twoactivities of BSF-1, induction of Ia expression on mouse B cells(Noelle, R. et al., Proc. Natl. Acad. Sci. U.S.A. 81:6149-6153 1984! andRoehm, N. et al., J. Exp. Med. 169:679-694 1984!; both of which areincorporated herein by reference) and enhancement of IgG₁ and IgEproduction as described above.

The COS supernatant had significant Ia inducing activity (FIG. 3C), andenhanced the secretion of both IgE and IgG₁, by LPS stimulated B cells(FIG. 3D). Results with this cDNA clone clearly show that all theseactivities are associated with a single gene product. An assay forstimulation of fibroblast growth using mouse 3T3 cells was negative.

H. Structure of the cDNA Insert for Clone 2A-E3

The cDNA insert was initially analyzed by restriction endonucleasedigestion; a restriction cleavage map of the cDNA insert and thestructure of the plasmid vector are shown in FIG. 2. The DNA sequence ofthe entire cDNA insert (FIG. A) was then determined using a standardcombination of Maxam-Gilbert chemical cleavage and dideoxy chaintermination methods well known to those in the art. The cDNA insert is585 base pairs long excluding the poly A tail. There is a single longopen reading frame, with the first ATG codon located 56 nucleotides fromthe 5' end followed by 140 codons ending with the termination codon TAGat nucleotide positions 476-478. The NH₂ -terminal segment of thepredicted polypeptide is hydrophobic, as would be expected for asecreted protein.

I. Expression of the Native Protein in T Cells and Homology to MouseIL-2 and IL-3

Assays of cell supernatants indicated that the expression of this geneproduct is inducible by Con A in Cl.Lyl⁺ 2⁻ /9 cells. Inducibleexpression of this gene was confirmed by analysis of mRNA isolated fromcells treated or untreated with Con A, which analysis indicated that asingle prominent mRNA species was detected only in mRNA isolated frominduced T-cells. mRNA samples from several mouse cell lines were thenanalyzed with the same radiolabelled probe. Hybridization was detectedin mRlqA from the EL-4 cell line treated with the phorbol ester, PMA.EL-4 is known to produce BSF-1 under these conditions. Two other linestested GK15-1 and LB2-1, represent a subset of T-cells which do notproduce combined MCGF/TCGF activities, and no hybridization with thelabelled probe was observed with the GK15-1 or LB2-1 mRNA samples. Theseresults show that there is good correlation between production ofbiological activities and expression of the mRNA.

Despite the biological activities of the polypeptides of the presentinvention that are similar to activities of IL-2 and IL-3, there is nosignificant nucleotide sequence homology between the mouse cDNA clonesof this invention and either mouse IL-2 or IL-3 cDNA sequences. At theamino acid sequence level, however, there are two regions which can bediscerned to have homology with these two gene products. Amino acidresidues 32-39 are 70% homologous to residues 49-56 of the IL-3precursor polypeptide (Yokota, T. et al., Proc. Natl. Acad. Sci. U.S.A.81:1070-1074 1984!). Amino acids 95-103 are 60% homologous with residues52-61 of mouse IL-2 (Yokota, T. et al., Proc. Natl. Acad. Sci. U.S.A.82:68-72 1985!). There are no other homologies which could be detectedwith other cloned lymphokines, such as gamma interferon (IFN) orinterleukin-1.

Both the biological activity data and analysis of mRNA levels suggestthat Cl.Lyl⁺ 2⁻ /9 cells produce high levels of this interleukin.Analysis of various T-cell clones suggests that only certain T-cellsexpress this gene product, and this subset often does not synthesizeIL-2 or gamma interferon.

EXAMPLE II

This example demonstrates the isolation of two cDNA clones containinggenes encoding polypeptides of the present invention that are active, inparticular, on human cells.

A. Human Cells

1) Human T Cell Line (2F1) and Peripheral Blood Lymphocytes (PBL)

A human helper T-cell clone, 2F1, and human peripheral blood lymphocytes(PBL's) were grown in Iscove's medium supplemented with 3% fetal calfserum. The 2F1 cells were activated with Con A (10 ug/ml) and PBL's werestimulated with 1 ng/ml PNA for 12 h, after which Con A at 5 ug/ml wasadded. The cells were harvested 4 hr (2F1) or 10 hr (PBL's) afteraddition of Con A.

2) Cell Suspensions

a) Preparation of human B cells

Enriched B cell preparations were isolated from human tonsils which wereobtained at tonsillectomy from patients with chronic tonsillitis. Tonsilcells were dispersed into single cell suspensions. The mononuclear cellswere isolated by Ficoll-Hypaque density gradient centrifugation(Pharmacia, Uppssala, Sweden) following the technique of Boyum, I.(1968) Scand. J. Clin. Lab. Invert. 21 (Suppl. 97) 77). B cells wererecovered after elimination of T-cells by rosetting with A.E.T. (2aminoethylisothiouronium bromide) treated sheep red blood cells. Therosetted cell mixture was layered over Ficoll Hypaque, and the rosetteforming cells were separated from the non-rosette forming cells bycentrifugation (20 min at 800 g).

After centrifugation, B cells were recovered at the interface and thecontaminating T-lymphocytes (5%) were further removed by a second cycleof rosetting. The B cell preparations contained>95% sIg⁺ cells asdetermined by staining with a fluorescein conjugated F(ab')2 fragmentgoat anti-human Ig; >95% human B cell specific antigen positive cells asdetermined by staining cells with a mouse anti-human B cell specificmonoclonal antibody: B1 (Coulter, Hialeh, Fla.) and fluoresceinconjugated F(ab')2 goat anti-mouse lg (Grub, Wien, Austria). Thecontamination with T-cells was less than 1% as determined by themonoclonal antibody Leu-1 (Becton Dickinson, Mountain View, Calif.) orthe monoclonal antibody OKT11 (Ortho, Raritan, N.J.). For activation,the B lymphocytes are resuspended in Yssel's culture medium (Yssel, H.et al. J. Immunological Methods 72:219(1984)) at 5×10⁵ per ml in avolume of 3 ml per well of a 6-well tissue culture plate (Falcon, Ref.3046, Oxnard, Calif.). The B- cells were cultured at 37° C. and in anatmosphere containing 5% CO₂.

b) Activation of human B cells

Two different activators were used:

i) Staphylococcus aureus strain Cowan I (SAC): SAC is obtained asPansorbin (Calbiochem, La Jolla, Calif.). It is added to the B cellcultures at a final concentration of 0.01%. Cells were activated bycultivation at 37° C. and 5% CO₂ for 24 hours, and were subsequentlycentrifuged over Ficoll Hypaque in order to remove non-viable cells andthe SAC particles.

ii) Anti IgM antibodies coupled to beads (Biorad, Richmond, Calif.): Thebeads are added at a final concentration of 5 ug anti IgM antibody perml of culture. Cells are activated by cultivation at 37° C. and 5% CO₂for 72 hours and subsequently isolated by centrifugation over FicollHypaque to remove the beads and the non-viable cells.

c) A human T-cell clone, JL-EBV, was stimulated with irradiated (4500R)cells of a human EBV-transformed B-cell line, and subsequentlymaintained in RPMI 1640 medium containing 10% human AB serum, 50 uM2-mercaptoethanol and recombinant human IL-2. Five to ten days afterstimulation, JL-EBV cells were used as targets in a two-day TCGF assay,using the colorimetric MTT method described previously.

3) Proliferation Assays

a) Assay of BCGF Activity

Cell proliferation was measured by ³ H-thymidine incorporation. Theactivated low density B cells are washed twice with culture medium andresuspended at 10⁶ per ml. 5×10⁴ B lymphocytes in 50 ul medium weredispensed into 96 well, flat bottomed microtiter trays (Falcon, Ref3072). 50 ul of supernatants to assay were added appropriately diluted.All assays were carried out in triplicate. After two days (anti μcultures), or three days (SAC cultures), the microcultures were pulsedwith 1 μCi tritiated thymidine (25 MCi/mmole, CEA, Saclay, France) perwell and were harvested 15 hours later using a MASH (MicrobiologicalAssociates, Bethesda, Md.). Dried glassfiber filters were counted in anLKB scintillation counter (LKB, Bromma, Sweden) after transfer intovials containing scintillation fluid (Ready Solv EP, Beckman, Fullerton,Calif.).

The B-cell growth factor activity of the tranfection supernatants wascompared to that of the following reagents: recombinant IL-2, commercialBCGF purified from peripheral blood cell culture supernatants stimulatedwith PEA (Cytokine Technology International, Buffalo, N.Y.), recombinanthuman gamma IFN, supernatant from a ConA stimulated human T-cell clonecontaining BCGF activity (HG 120).

b) Assay of TCGF Activity

The human helper T-cell clone JL-EBV was stimulated with irradiated(4500R) cells of a human EBV-transformed B-cell line, and subsequentlymaintained in RPMI 1640 medium containing 10% human AB serum, 50 uM2-mercaptoethanol (2ME) and recombinant human IL-2. Human PBL's werestimulated with PEA (20ug/ml) and maintained in RPMI 1640 containing 10%FBS, 50 uM 2ME and recombinant human IL-2. Five to ten days afterstimulation, JL-EBV cells or PEA blasts were used as targets in atwo-day TCGF assay, using the colorimetric MTT method describedpreviously or in a three-day TCGF assay, using ³ H!thymidineincorporation.

B. mRNA Isolation: pcD Library Construction; and DNA Hybridizations; andDNA Transfections.

All of the procedures were carried out essentially as described inExample I above.

pcD cDNA libraries were constructed with mRNA from ConA-induced 2F1cells and peripheral blood lymphocytes by using a modified pcDV1 plasmidcontaining an NsiI site at the previous location of the KpnI site (Lee,F. et al. Proc. Natl. Acad. Sci. U.S.A. 82: 4360-4364 1985!), which isincorporated herein by reference. Each of the DNA libraries contained aminimum of 5×10⁵ independent clones.

A PstI fragment was isolated from a mouse 2A-E3 cDNA clone, labeled bynick translation (1×10 ⁸ cpm/μg) and used to probe nitrocellulosefilters containing plasmid DNA preparations from ten pools, eachrepresenting approximately 1×10³ clones of 2F1 cDNA library. Lowstringency hybridization conditions (overnight at 42° C.) were used:6×SSPE (1×SSPE=180 mM NaCl/10 mM sodium phosphate, pH 7.4/1 mM EDTA)(Maniatis, T. et al. "Molecular Cloning: A Laboratory Manual", ColdSpring Harber Laboratory, New York 1982!), 20% (vol/vol) formamide, 0.1%sodium dodecyl sulfate, yeast carrier tRNA at 100 μl. The filters werewashed with 2×SSPE, 0.1% sodium dodecyl sulfate at 37° C.

C. Identification and Analysis of Hybridizing Human cDNA Clones.

This positive pool was used in further colony filter hybridizations toidentify a single clone that hybridized with the mouse probe (clone#46).

After making a restriction endonuclease cleavage map of clone #46, twohuman peripheral blood lymphocyte pcD libraries were screened, using anNheI-EcoRI fragment as probe, under stringent conditions. Five cDNAclones were identified out of 1×10⁵ clones screened in one library andone cDNA clone out of 1×10⁴ clones from the second. Analysis withrestriction endonucleases showed that each of the hybridizing clones isidentical in structure to the 2F1-derived cDNA clone #46 (pcD-46).

The DNA sequence of the cDNA insert of clone #46 was determined and isshown in FIG. 1B. The cDNA insert is 615 bp long, excluding the poly(A)tail. There is a single open reading frame, with the first ATG codonlocated at 64 nucleotides from the 5' end followed by 153 codons endingwith the termination codon TAG at nucleotide positions 523-525. TheNH2-terminal segment of the predicted polypeptide is hydrophobic, aswould be expected for a secreted protein.

A comparison between the coding regions of a human and a mouse cDNA ofthe present invention revealed that the regions of the human cDNA codingsequence in pcD-46 covered by amino acid positions 1-90 and 129-149share approximately 50% homology with the corresponding regions of themouse cDNA (2A-E3) coding sequence. These regions, and 5' and 3'untranslated regions, share about 70% homology between the two cDNAsequences from the different species, whereas the region covered byamino acids 91-128 of the human protein shares very limited homologywith the corresponding mouse region. In all, six of the seven cysteineresidues in the human protein are conserved in the related mouseprotein. Some amino acid sequence homology exists between a native formof a human polypeptide of the present invention and mouse IL-3. Aminoacid residues 7-16 and 120-127 are 50% and 55% homologous, respectively,to residues 16-27 and 41-49 of the mouse IL-3 precursor polypeptide(Yokota, T. et al., Proc. Natl. Acad. Sci. U.S.A. 81:1070-1074 1984!).

D. Some Biological Activities of Polypeptides Encoded by Human cDNAClones.

Supernatants from COS cell cultures transfected with the pcD vectorcontaining human cDNA #46 were tested initially for activity onPEA-stimulated human peripheral blood cells, and positive effects wereseen. A human T-cell line, JL-EBV that undergoes IL-2 dependent growthafter antigen stimulation was utilized for TCGF activity studies. FIG. 4shows that both T-cell populations responded strongly to human IL-2 andalso responded at a lower level to supernatants from COS cellstransfected with a human cDNA of the present invention. Whether measuredby ³ H!thymidine uptake or the MTT colorimetric assay, the slopeproduced by the polypeptides from the transfected COS cells responsecurve was less than that of IL-2, and the saturation level ofstimulation was half or less of the level seen in response to IL-2.

The original cDNA clone #46 gave a very low titer, and this was improvedby expressing the same cDNA clone in L-cells, or by deleting the 36residue long oligo(dG) stretch in the original clone to produce pcD-125.The vector pcD-125 was formed as follows: pcD-46 was cleaved with Sau3Ato isolate a fragment containing the 5' 162 nucleotides of the cDNAinsert (eliminating the GC segment) and then the fragment was insertedinto the BglII site of p101. The plasmid p101 was derived from pcD-mouseIL-3 (see, Yokota, T. et al. 1984!above) and is deleted for the sequencefrom the PstI site at the 5' end of the cDNA to a BglII site within themouse IL-3 cDNA. A BglII site is included at the junction of the deletedsequence. The Sau3A fragment is fused to the SV40 promoter as in pcD-46,except for the GC stretch. The remainder of the human cDNA was thenreconstructed with a HindIII--NheI fragment from pcD-46 which carriesthe 3' end of the cDNA, the SV40 poly A site and all of the pBR322sequences of pcD-46.

A high expression vector (pEBT178) can be constructed with the plasmidpcD4-RSV-IL3, which carries the mouse IL-3 cDNA in the pcD vectormodified in the following way. The SV40 origin fragment is in thereverse orientation, i.e., the late promoter, rather than the earlypromoter, is in the same orientation as the mouse IL-3 cDNA. A 580bpHindIII-XhoI fragment of the Rous sarcoma virus (RSV) promoter (Gorman,C. et al., Proc. Natl. Acad. Sci. U.S.A. 79:6777-6781 1982!, which isincorporated herein by reference) can be isolated from a variety ofsources (e.g., pRSVcat or pRSV-βglobin) which can be modified byconverting the HindIII site at the 3' junction of the RSV LTR region toan XhoI site and an upstream NdeI site to an HindIII site. This RSVpromoter can be inserted between the SV40 origin and the SV96 splicejunctions such that the RSV promoter could transcribe the downstreamcDNA. Unique AatII and NdeI sites in this resulting plasmid can beconverted to SalI fragment carrying the SV40 origin, the RSV promoterand the cDNA. This SalI fragment can then be inserted into plasmid p201(Yates, J., et al., Nature 313:812-815 1985!, which is incorporatedherein by reference) at the location of the unique ClaI site, which hadbeen converted to a SalI site. The IL-3 cDNA can then be removed bycleaving with XhoI and replaced with the corresponding XhoI fragmentcontaining the human cDNA isolated from pcD-125. The results usingpcD-125 suggest that the oligo(dG) segment in pcD-46 is inhibitory toexpression of the downstream cDNA insert.

For the following experiments, the human cDNA clone #125 was transfectedinto COS cells twice, and supernatants obtained (B59-4-125 andB59-5-132). A mock supernatant was prepared with an irrelevant cDNAclone, as described previously.

a) SAC Blast Assay

As shown in Table I, the SAC preactivated B cells were found toproliferate in response to recombinant IL-2 (produced in E. Coli),commercial BCGF (purified from PBL culture stimulated with PHA, obtainedfrom Cytokine Technology International, Buffalo N.Y.). and a T-cellclone (HG-120) supernatant. The transfection supernatants did notsignificantly stimulate proliferation as measured by thymidineincorporation. These transfection supernatants neither enhanced norinhibited the proliferation induced by optimal commercial BCGFconcentrations.

                                      TABLE I                                     __________________________________________________________________________    ACTIVITY OF THE CLONE #125 TRANSFECTION SUPERNATANTS                          ON SAC PREACTIVATED B CELLS                                                          Transfection Supernatant Concentration (% v/v)                                0      0.01  0.04   0.2    1      5      15                                   .sup.3 HTdR incorporation (c.p.m. ≠ SD)                          __________________________________________________________________________    Medium 2237 ± 487                                                          MOCK   2237 ± 487                                                                        1079 ± 67                                                                        1564 ± 395                                                                        1789 ± 313                                                                         40 ± 72                                                                          1285 ± 62                                                                         1560 ± 289                 MOCK + 12992 ± 2759                                                                      n.d.  15684 ± 1622                                                                      13126 ± 2887                                                                      13714 ± 2014                                                                      5848 ± 859                                                                        10128 ± 1703               BCGFc 10%                                                                     B 59-4-125                                                                           2237 ± 487                                                                        2086 ± 429                                                                        861 ± 377                                                                         2682 ± 1128                                                                      2374 ± 179                                                                        2826 ± 526                                                                        4701 ± 242                 B 59-4-125 +                                                                         12992 ± 2789                                                                      n.d.  12655 ± 697                                                                       5655 ± 280                                                                        6765 ± 825                                                                        10023 ± 149                                                                       10924 ± 506                BCGFc 10%                                                                     B 59-5-132                                                                           2237 ± 487                                                                        1424 ± 162                                                                       1328 ± 185                                                                        1999 ± 56                                                                         2272 ± 309                                                                        3493 ± 796                                                                        3870 ± 668                 B 59-5-132 +                                                                         12992 ± 2789                                                                      n.d.  12631 ± 492                                                                        7203 ± 3212                                                                      7567 ± 895                                                                        10119 ± 584                                                                       12277 ± 1014               BCGFc 10%                                                                     __________________________________________________________________________     Controls:                                                                     BCGFc (25%): 15869 ± 1039                                                  Gamma IFN 5000 U/ml: 3461 ± 832                                            IL2 10 U/ml: 18371 ± 644                                                   ConA 10 μg/ml: 254 ± 62                                                 PHA 1%: 660 ± 38                                                           Supernatant of clone HG120 (25%) activated by ConA (10 μg/ml): 8340        ± 784                                                                 

b) Anti μ Blast Activity

The anti μ preactivated B cells were found to proliferate moderately inresponse to recombinant IL-2 and commercial BCGF, whereas they stronglyproliferated in response to the T-cell clone supernatant and theB-59-4-125 and B-59-5-132 transfection supernatants. The transfectionsupernatants induced proliferation at concentrations as low as 1% (see,Table II).

                  TABLE II                                                        ______________________________________                                        BCGF ACTIVITY OF THE CLONE #125 TRANSFECTION                                  SUPERNATANTS ON ANTI-μ PREACTIVATED B CELLS                                % (v/v) of                                                                    supernatants                                                                            3HTdR incorporation (c.p.m. = SD)                                   added     MOCK        B 59-4 125                                                                              B 59-5 132                                    ______________________________________                                        0          278 ± 163                                                                              278 ± 163                                                                            278 ± 163                                 0.2       189 ± 64 144 ± 65                                                                             157 ± 28                                   1         323 ± 44 1313 ± 227                                                                           1078 ± 71                                  5         408 ± 59 4314 ± 231                                                                            3762 ± 1097                               25        397 ± 74 N.D.      4289 ± 369                                 ______________________________________                                         Positive controls:                                                            BCGF (25%): 1710 ± 123                                                     Supernatant (25%) of Tcell clone HG120 activated by ConA (10 μg/ml):       3559 ± 138                                                                 IL2 (10 U/ml) = 1502 ± 414                                            

When tested in combination with optimal concentrations of commercialBCGF, these two transfection supernatants had additional proliferativecapacity (see, Table III). The MOCK transfection supernatant had neitherstimulatory nor inhibitory activity on the anti μ preactivated B cells.

                  TABLE III                                                       ______________________________________                                        THE COMBINED EFFECTS OF COMMERCIAL BCGF AND THE                               CLONE #125 TRANSFECTION SUPERNATANTS ON ANTI-μ                             PREACTIVIATED B CELLS                                                         % (v/v)      3HTdR incorporation (c.p.m. = SD)                                SN added     MOCK      B 59-4 125 B 59-5 132                                  ______________________________________                                        0       --        346 ± 116                                                                            346 ± 116                                                                           346 ± 116                             BCGF 10%                                                                              --       1835 ± 651                                                                           1835 ± 651                                                                          1835 ± 651                             "       0.04     1660 ± 304                                                                           1756 ± 498                                                                          1911 ± 200                             "       0.2      1362 ± 257                                                                           2303 ± 224                                                                          2713 ± 158                             "       1        1699 ± 160                                                                           3784 ± 171                                                                          3507 ± 316                             "       5        1518 ± 246                                                                           7921 ± 217                                                                           7463 ± 1508                           "       15       1093 ± 272                                                                            8487 ± 1042                                                                         8389 ± 1060                           ______________________________________                                         Positive control:                                                             BCGF (25%): 2049 ± 222                                                

c) The effects of the supernatants of COS cells transfected with thehuman cDNA clone #125 on proliferation of human B cells preactivated bybeads coated with anti-μ antibodies

The test was carried out as described above. Briefly, the B cells (>95%pure) were obtained from tonsils and purified by depletion of theT-cells by two rounds of rosetting with AET-treated sheep erythrocytes.These B cells were preactivated by anti-μ antibodies coupled to beads byincubation at 37° C. and 5% CO₂ for 24 and 72 hours respectively. Afterthis activation period, the beads were removed by centrifugation overFicoll-Hypaque and the B cells were washed twice and then seeded atconcentrations of 5×10⁴ /flat bottom 0.2 ml well. Transfectionsupernatants were added at various dilutions. In addition, theactivities of commercially available BCGF (25% v/v) and recombinant IL-2(200 IU/ml) were tested. The B cells preactivated by 24 hours werecultured for 4 days in the presence of the test supernatants (totalincubation period 5 days). The B cells preactivated for 72 hours werecultured for 3 days in the presence of the test supernatants (totalincubation period 6 days).

The results, shown in Table IV, indicate that the transfectionsupernatants B-50-4125 and B-59-5132 act both on B cells preactivatedfor 24 hours and 72 hours, respectively. Significant BCGF activity wasobserved at concentrations of 0.2% and increased at higherconcentrations. Furthermore, commercial BCGF, the supernatant of HG-120,as well as recombinant IL-2, were all found to have BCGF activity.

                                      TABLE IV                                    __________________________________________________________________________    THE EFFECTS OF SUPERNATANTS OF COS7 CELLS TRANSFECTED                         WITH CLONE #125 ON B CELLS PREACTIVATED                                       WITH ANTI-μ ANTIBODIES FOR 24 HRS AND 72 HRS RESPECTIVELY                  % (v/v)                                                                             B cells preactivated for 24 hrs                                                                   B cells preactivated for 72 hrs                     supernatant                                                                         MOCK  B 59-4125                                                                            B 59-5132                                                                            MOCK B 59-4125                                                                            B 59-5132                               added 3HtdR Incorp. c.p.m. ± S.D.                                                                    3HTdR Incorp. c.p.m. ± S.D.                      __________________________________________________________________________    0           1096 ± 139      235 ± 67                                    0.2   913 ± 180                                                                        3817 ± 187                                                                        4147 ± 337                                                                        149 ± 38                                                                         546 ± 127                                                                        419 ± 13                             1     900 ± 117                                                                        5590 ± 392                                                                        5524 ± 379                                                                        213 ± 80                                                                        1790 ± 106                                                                        1686 ± 117                           5     799 ± 153                                                                        10544 ± 329                                                                       11154 ± 1145                                                                      148 ± 38                                                                        2409 ± 181                                                                        1931 ± 478                           15    1032 ± 296                                                                       11600 ± 872                                                                       14486 ± 1827                                                                      330 ± 90                                                                        2563 ± 583                                                                        2691 ± 451                           25    2022 ± 1256                                                                      14687 ± 1133                                                                      15277 ± 776                                                                       445 ± 2                                                                          3863 ± 1473                                                                      2979 ± 43                            commercial BCGF (25% v/v): 3545 ± 324                                                                commercial BCGF (25% v/v): 1193 ± 55             supernatant HG-120 (25% v/v): 12451 ± 1273                                                           sup. HG-120 (25% v/v): 3091 ± 266                rec. IL-2 (10 U/ml): 45331 ± 1622                                                                    IL-2 (10 U/ml): 6997 ± 524                       __________________________________________________________________________

These results indicate that some supernatants from COS cells transfectedwith plasmids harboring a gene of the present invention induce theproliferation of human B cells preactivated with optimal concentrationsof anti-IgM antibodies coupled to beads. These supernatants had additiveproliferative capacity with commercial BCGF and do not significantlyinduce the proliferation of SAC preactivated human B cells. Thecommercial BCGF preparation contains factors which are different fromthe factors of the present invention. The data also suggest thatdifferent B cell subpopulations or B cells in different stages ofactivation/differentiation respond to different BCGF's.

d) The effects of the supernatants of COS cells tranfected with thehuman cDNA clone #125 on the proliferation of human B cells preactivatedby SAC for 3 days

We investigated whether B cells preactivated with SAC for 72 hours couldbe induced to proliferate by the human cDNA clone #125 transfectionsupernatants. The experiment was carried out as described previouslywith the exception that the B cells were preactivated for 72 hours. InTable V it is shown that B cells preactivated by SAC for 72 hours andcultured for an additional 3 days with the test supernatants do respond.At concentrations of 0.2%, significant proliferation is induced, whichincreased at higher concentrations of supernatant. Commercial BCGF, thesupernatant of T-cell clone HG-120 and recombinant Il-2 are also activein this assay.

                  TABLE V                                                         ______________________________________                                        THE EFFECTS OF SUPERNATANTS OF COS CELLS                                      TRANSFECTED WITH CLONE #125 ON B CELLS                                        PREACTIVATED BY SAC FOR 72 HRS                                                B cells preactivated                                                          % (v/v)   3HTdR incorp. (c.p.m. ± S.D.)                                    supernatant added                                                                       MOCK       B 59-4125   B 59-5132                                    ______________________________________                                        0                    1621 ± 503                                            0.2       1361 ± 377                                                                            2922 ± 451                                                                             2874 ± 544                                1         1107 ± 250                                                                            4387 ± 461                                                                             4544 ± 408                                5         1076 ± 104                                                                            7480 ± 739                                                                             6933 ± 693                                15         925 ± 149                                                                            7874 ± 298                                                                             7422 ± 872                                25        1316 ± 250                                                                             8532 ± 1414                                                                            9998 ± 1064                              ______________________________________                                         commercial BCGF (25% v/v): 6744 ± 181                                      sup. HG120 (25% v/v): 1935 ± 3089                                          rec. IL2 (10 U/ml): 17509 ± 1406                                      

e) The effects of supernatants of COS cells transfected with the humancDNA clone #125 non-preactivated human B cells

In order to determine whether the transfection supernatants B-59-4-125and B-59-4-132 had B cell activation activity, their effects were testedon non-preactivated human B cells purified from tonsils.

1×10⁵ purified B cells (>95% pure) were seeded per 0.2 ml flat bottomwell and incubated for 5 days in the presence of the transfectionsupernatants at various dilutions. The results are shown in Table VI.

                                      TABLE VI                                    __________________________________________________________________________    THE EFFECTS OF SUPERNATANTS OF COS CELLS TRANSFECTED                          WITH CLONE #125 ON NON-PREACTIVATED HUMAN B CELLS                             Source of                                                                           % (v/v) supernatant added                                               Supernatant                                                                         0     0.04  0.2   1     5     15                                        __________________________________________________________________________    Medium                                                                              1685 ± 511                                                           Mock        1388 ± 85                                                                        1032 ± 139                                                                       1126 ± 141                                                                        979 ± 216                                                                       653 ± 198                              B 59-4-125   912 ± 16                                                                        889 ± 104                                                                        1081 ± 32                                                                        1834 ± 223                                                                       1280 ± 37                              B 59-4-132  1250 ± 68                                                                        929 ± 130                                                                        1283 ± 124                                                                       1972 ± 104                                                                       1620 ± 270                             __________________________________________________________________________     BCGFc 25%: 2031 ± 572                                                      PHA 1%: 509 ± 270                                                          Cowan: 60797 ± 2481                                                   

The transfection supernatants were not effective in inducingnon-preactivated resting human B cell proliferation, indicating thatthey do not contain B cell activation activity. In addition, commercialBCGF was not effective in the system. The purified B cells respondedwell to activation by SAC indicating that the negative results with thetransfection supernatants could not be attributed to intrinsicnon-responsiveness of the B cell preparation.

The results presented here indicate that B cells preactivated withanti-μ for 24 hours in contrast to B cells preactivated with SAC for 24hours (as shown previously) showed proliferative responses to the clone#125 transfection supernatants. Furthermore, it is shown that B cellspreactivated for 72 hours by SAC also can be induced to proliferate inresponse to the transfection supernatants. These results suggest thatthe B cells may have to reach a certain differentiation/activation stagebefore becoming fully responsive to some polypeptides to the presentinvention. The human clone #125 gene product does not induce theproliferation of non-preactivated human B cells in this system.

As shown, supernatants from COS cells transfected with plasmids bearinga human cDNA of the present invention induce the proliferation of normalhuman T-cells and a human T-cell clone, an activity which is similar tohuman IL-2. However, in the in vitro assays conducted, the maximumextent of proliferation of human T-cells induced in response to thenative polypeptides is about half of that induced by human IL-2. Theproliferation inducing activity of the polypeptides could not beinhibited by monoclonal antibodies against IL-2 or the IL-2 receptor.These results suggest that the polypeptides can act directly on T-cells(not through the induction of IL-2) and that their activity is notnecessarily mediated by the IL-2 receptor. The transfection supernatantsalso stimulate the proliferation of human B-cells preactivated withoptimal concentrations of anti-IgM antibodies coupled to beads and haveadditive proliferative capacity with a commercial BCGF preparation.

This additive capability, which is typically species specific, isevident when the polypeptides of the present invention are used withother immune-reactive agents; i.e., other agents capable of exerting aresponse on cells of the immune network. By way of example and notlimitation, such polypeptides can exhibit the following activities: (a)minimal effects on colony forming cells (e.g., promotes survival withoutproliferation) unless other immune-reactive agents are present; (b) inthe presence of mouse IL-3, which induces from mouse bone marrowproliferation of some eosinophils, mast cells, macrophages, neutrophilsand bursts (colonies of cells containing precursors to erythrocytes,among other cells), the numbers of eosinophils, mast cells, neutrophils,and bursts (size as well) are greatly increased while the number andsize of macrophages are decreased; (c) in the presence of mouse G/M-CSF,which induces from mouse bone marrow proliferation of neutrophils,macrophages, eosinophils and some bursts, the numbers of all cell typesare greatly increased, except macrophages; (d) in the presence of humanG/M-CSF, which induces proliferation from human cord blood ofneutrophils, macrophages and eosinophils, macrophage proliferation isdiminished, the numbers of neutrophils and eosinophils are increased,and basophils appear in significant amounts; (e) in the presence ofmouse G-CSF, which induces proliferation from mouse bone marrow ofgranulocytes (particularly neutrophils), more and larger colonies ofgranulocytes are produced; and (f) in the presence of mouse macrophagecolony stimulating factor (M-CSF), which induces proliferation frommouse bone marrow of macrophages, less and smaller colonies ofmacrophages are produced, but the macrophage colonies remaining appearto remain viable.

Thus some of the polypeptides of the present invention are capable ofaugmenting the activity of various CSF's on progenitor and committedcell lineages, without always altering each of the CSF's activites. Thisaugmenting is typically synergistic, and may only become evident whenother immune-reactive agents (e.g., anti-IgM) are also present. Thepolypeptides can induce the initial proliferation of some hematopoieticcells, but such proliferation may require additional factors tocontinue.

From the foregoing, it will be appreciated that the cDNA clones of thepresent invention provide accurate and complete sequence data on amammalian lymphokine. The invention also provides to those skilled inthe art means for producing significant quantities of the factor(essentially free from other hematopoietic factors) for the improved invitro maintenance of T-cells, granulocytes and mast cells, as well asstimulation of other hematopoietic cells (e.g., B cells, macrophages,etc.). Further, the information gleaned from the cDNA clones increasesunderstanding of the mammalian immune response, enhancing experimentalresearch and therapeutic capabilities.

Although the invention has been described in some detail by way ofexample for purposes of clarity and understanding, it will be readilyapparent to those skilled in the art that certain changes andmodifications may be practiced within the scope of the appended claims.

We claim:
 1. A pharmaceutical composition comprising a pharmaceuticallycompatible carrier and a therapeutically effective amount of humaninterleukin-4.
 2. The composition of claim 1, wherein said humaninterleukin-4 comprises the sequence:

    __________________________________________________________________________    His                                                                              Lys                                                                              Cys                                                                              Asp                                                                              Ile                                                                              Thr                                                                              Leu                                                                              Gln                                                                              Glu                                                                              Ile                                                                              Ile                                                                              Lys                                                                              Thr                                                                              Leu                                                                              Asn                                                                              Ser                              Leu                                                                              Thr                                                                              Glu                                                                              Gln                                                                              Lys                                                                              Thr                                                                              Leu                                                                              Cys                                                                              Thr                                                                              Glu                                                                              Leu                                                                              Thr                                                                              Val                                                                              Thr                                                                              Asp                                                                              Ile                              Phe                                                                              Ala                                                                              Ala                                                                              Ser                                                                              Lys                                                                              Asn                                                                              Thr                                                                              Thr                                                                              Glu                                                                              Lys                                                                              Glu                                                                              Thr                                                                              Phe                                                                              Cys                                                                              Arg                                                                              Ala                              Ala                                                                              Thr                                                                              Val                                                                              Leu                                                                              Arg                                                                              Gln                                                                              Phe                                                                              Tyr                                                                              Ser                                                                              His                                                                              His                                                                              Glu                                                                              Lys                                                                              Asp                                                                              Thr                                                                              Arg                              Cys                                                                              Leu                                                                              Gly                                                                              Ala                                                                              Thr                                                                              Ala                                                                              Gln                                                                              Gln                                                                              Phe                                                                              His                                                                              Arg                                                                              His                                                                              Lys                                                                              Gln                                                                              Leu                                                                              Ile                              Arg                                                                              Phe                                                                              Leu                                                                              Lys                                                                              Arg                                                                              Leu                                                                              Asp                                                                              Arg                                                                              Asn                                                                              Leu                                                                              Trp                                                                              Gly                                                                              Leu                                                                              Ala                                                                              Gly                                                                              Leu                              Asn                                                                              Ser                                                                              Cys                                                                              Pro                                                                              Val                                                                              Lys                                                                              Glu                                                                              Ala                                                                              Asn                                                                              Gln                                                                              Ser                                                                              Thr                                                                              Leu                                                                              Glu                                                                              Asn                                                                              Phe                              Leu                                                                              Glu                                                                              Arg                                                                              Leu                                                                              Lys                                                                              Thr                                                                              Ile                                                                              Met                                                                              Arg                                                                              Glu                                                                              Lys                                                                              Tyr                                                                              Ser                                                                              Lys                                                                              Cys                                                                              Ser                              Ser.                                                                          __________________________________________________________________________


3. The composition of claim 1, wherein said human interleukin-4 has amolecular weight of about 15,000 daltons.
 4. The composition of claim 1,wherein said interleukin-4 is chemically synthesized.
 5. The compositionof claim 1, wherein said interleukin-4 is recombinantly produced.
 6. Thecomposition of claim 5, wherein said interleukin-4 exhibits an activityselected from:a) T cell growth factor activity; b) mast cell growthfactor activity; c) Ia antigen activity; and d) IgE and IgG1 enhancingactivity.
 7. The composition of claim 6, wherein said interleukin-4exhibits a plurality of said activities.
 8. The composition of claim 5,wherein said interleukin-4 is encoded by a nucleic acid constructcomprising a heterologous signal sequence.
 9. The composition of claim5, wherein said interleukin-4 is recombinantly produced in:a) abacterial cell; b) a yeast cell; or c) a mammalian cell.
 10. Thecomposition of claim 1, wherein said interleukin-4 is glycosylated. 11.The composition of claim 1, in a unit dose of interleukin-4.
 12. Thecomposition of claim 11, wherein said unit dose is in range of 1 μg to100 mg.
 13. The composition of claim 1, suitable for parenteraladministration.
 14. The composition of claim 11, wherein saidinterleukin-4 exhibits an activity selected from:a) T cell growth factoractivity; b) mast cell growth factor activity; c) Ia antigen activity;and d) IgE and IgG1 enhancing activity.
 15. The composition of claim 14,wherein said interleukin-4 exhibits a plurality of said activities.